Antibodies that potently neutralize hepatitis B virus and uses thereof

ABSTRACT

The present invention relates to antibodies, and antigen binding fragments thereof, that bind to the antigenic loop region of hepatitis B surface antigen (HBsAg) and potently neutralize infection of both hepatitis B virus (HBV) and hepatitis delta virus (HDV). The invention also relates to epitopes to which the antibodies and antigen binding fragments bind, as well as to nucleic acids that encode and cells that produce such antibodies and antibody fragments. In addition, the invention relates to the use of the antibodies and antibody fragments of the invention in the diagnosis, prophylaxis and treatment of hepatitis B and hepatitis D.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is 470085_405C1_SEQUENCE_LISTING.txt. The text fileis 46.8 KB, was created on Apr. 30, 2020, and is being submittedelectronically via EFS-Web.

The present invention relates to the field of antibodies againsthepatitis B virus (HBV) and against hepatitis delta virus (HDV) and usesthereof. The potent anti hepatitis B antibodies of the present inventionbind to an epitope located in the antigenic loop region of the S domainof the HBV envelope proteins (HBsAg), as identified in the presentinvention. The invention also relates to nucleic acids that encode andimmortalized B cells and cultured plasma cells that produce suchantibodies and antibody fragments. In addition, the invention relates tothe use of the antibodies and antibody fragments of the invention inscreening methods as well as in the diagnosis, prophylaxis and treatmentof diseases, in particular hepatitis B and hepatitis D.

The hepatitis B virus (HBV) consists of (i) an envelope containing threerelated surface proteins (hepatitis B surface antigen, HBsAg) and lipidand (ii) an icosahedral nucleocapsid enclosing the viral DNA genome andDNA polymerase. The HBV capsid is formed in the cytosol of the infectedcell during packaging of an RNA pregenome replication complex and gainsthe ability to bud during synthesis of the viral DNA genome by reversetranscription of the pregenome in the lumen of the particle. The threeHBV envelope proteins S-HBsAg, M-HBsAg, and L-HBsAg shape a complextransmembrane fold at the endoplasmic reticulum, and formdisulfide-linked homo- and heterodimers. During budding at anintracellular membrane, a short linear domain in the cytosolic preSregion interacts with binding sites on the capsid surface. The virionsare subsequently secreted into the blood. In addition, the surfaceproteins can bud in the absence of capsids and form subviral particles(SVP's) which are also secreted in 3-4 log excess over virions. Highlevel of HBsAg can exhaust HBsAg-specific T-cell response, and isproposed as an important factor for viral immunotolerance in patientswith chronic hepatitis B (CHB) (Chisari F V, Isogawa M, Wieland S F,Pathologie Biologie, 2010; 58:258-66).

Hepatitis B virus causes potentially life-threatening acute and chronicliver infections. Acute hepatitis B is characterized by viremia, with orwithout symptoms, with the risk of fulminant hepatitis occurrence (LiangT J, Block T M, McMahon B J, Ghany M G, Urban S, Guo J T, Locarnini S,Zoulim F, Chang K M, Lok A S. Present and future therapies of hepatitisB: From discovery to cure. Hepatology. 2015 Aug. 3. doi:10.1002/hep.28025. [Epub ahead of print]). Despite an efficaciousvaccine against hepatitis B is available since 1982, WHO reports that240 million people are chronically infected with hepatitis B and morethan 780 000 people die every year due to hepatitis B complications.Approximately one third of chronic hepatitis B (CHB) patients developcirrhosis, liver failure and hepatocellular carcinoma, accounting for600,000 deaths per year (Liang T J, Block T M, McMahon B J, Ghany M G,Urban S, Guo J T, Locarnini S, Zoulim F, Chang K M, Lok A S. Present andfuture therapies of hepatitis B: From discovery to cure. Hepatology.2015 Aug. 3. doi: 10.1002/hep.28025. [Epub ahead of print]).

The currently available treatments for chronic hepatitis B include(pegylated) interferon-alpha (IFN-α or pegIFN-α) and nucleos(t)ideanalogue Direct Acting Antivirals (DAAs) that inhibit the hepatitis Bvirus (HBV) DNA polymerase (“polymerase inhibitors”). Polymeraseinhibitors include Lamivudine, Adefovir, Entcavir, Telbivudine andTenofovir. Polymerase inhibitors (Lamivudine, Adefovir, Entecavir,Telbivudine, Tenofovir) suppress the reverse transcriptase function ofthe HBV DNA polymerase and therefore interfere with the synthesis ofviral DNA from pregenomic RNA. This treatment does not prevent viralspread, formation of cccDNA and does not affect HBsAg release. Moreover,polymerase inhibitors limit disease progression but rarely clear thevirus. Thus, viral relapse is commonly observed after stopping thetreatment and, therefore, polymerase inhibitors should be used for thelifetime. In addition, drug-resistant mutants emerge after prolongedtreatment. PEG-IFN-α inhibits HBV indirectly through immune modulatoryeffects and directly by reducing steady-state levels of HBV transcripts(increased acetylation of cccDNA-bound histones). However, PEG-IFN-α haslimited efficacy and causes serious side effects.

While pegIFN-α is effective in approximately one-third of the treatedpatients, the polymerase inhibitors significantly reduce viral load inthe vast majority of those treated (Timothy M. Block, Robert Gish,Haitao Guo, Anand Mehta, Andrea Cuconati, W. Thomas London, Ju-Tao GuoChronic hepatitis B: What should be the goal for new therapies?Antiviral Research 98 (2013) 27-34). Interferon α is associated withmany adverse reactions and cannot be used in patients with advancedcirrhosis or medical/psychiatric contraindications (Liang T J, Block TM, McMahon B J, Ghany M G, Urban S, Guo J T, Locarnini S, Zoulim F,Chang K M, Lok A S. Present and future therapies of hepatitis B: Fromdiscovery to cure. Hepatology. 2015 Aug. 3. doi: 10.1002/hep.28025.[Epub ahead of print]). Although polymerase inhibitors such asentecarvir and tenofovir appear to have less adverse effects, rates ofHBeAG seroconversion and HBsAg loss are low for those drugs. Therefore,most patients require often lifelong treatment with associated costs andrisks of adverse reactions, drug resistance and non-adherence. Thus, thecurrently available treatment for chronic hepatitis B is stillhandicapped by various limitations and cannot be considered as curative.Therefore—although treatment for HBV has improved—HBV patients oftenrequire life-long therapies and cure is still a challenging goal. (LiangT J, Block T M, McMahon B J, Ghany M G, Urban S, Guo J T, Locarnini S,Zoulim F, Chang K M, Lok A S. Present and future therapies of hepatitisB: From discovery to cure. Hepatology. 2015 Aug. 3. doi:10.1002/hep.28025. [Epub ahead of print]). The closest outcome to curechronic hepatitis B (CHB) and an ideal endpoint of treatment would be toachieve a loss of hepatitis B surface antigen (HBsAg), which is,however, not yet achieved efficiently with the presently availabletreatments of chronic hepatitis B (for review see Gish R. G. et al.,2015, Antiviral Research 121:47-58).

Severely decompensated HBV patients with acute hepatitis orhepatocellular carcinoma are indicated for orthotopic livertransplantation (OLT). After OLT, the hepatitis B recurrence rateis >80% without prevention, while >90% of transplant recipients areclinically controlled with combined hepatitis B immunoglobulin (HBIG)and nucleos(t)ide analogue treatment. Hepatitis B immunoglobulins (HBIG)are polyclonal immunoglobulins purified from vaccinated donors. However,long-term HBIG administration is associated with several unresolvedissues, including limited availability and extremely high cost (TakakiA, Yasunaka T, Yagi T. Molecular mechanism to controlpost-transplantation hepatitis B recurrence. Int J Mol Sci. 2015 Jul.30; 16(8):17494-513). Moreover, extremely high doses have to beadministered, namely 10 grams (containing 10,000 IU based on bindingassays) are administered to the recipient during the transplant byintravenous infusion. Subsequently 2 grams are administeredintravenously daily for 8 days and further infusions are given every 1-3months to maintain anti-HBs serum levels above 100 IU/ml. Again,life-long treatment is required.

Even more severe complications are observed when coinfection orsuperinfection with hepatitis delta virus (HDV) occur. According to theWHO, hepatitis D infects about 15 million people worldwide. HDV isconsidered a subviral satellite because it can propagate only in thepresence of HBV. HDV is one of the smallest animal viruses (40 nm),whereby its genome is only 1.6 kb and encodes for S and L HDAg. Allother proteins needed for genome replication of HDV, including the RNApolymerase, are provided by the host cell and the HDV envelope isprovided by HBV. In other words, HDV is a defective virus that requirescoinfection with HBV for its replication since it utilizes the hepatitisB envelope proteins (HBsAg) as its own virion coat. When introduced intopermissive cells, the HDV RNA genome replicates and associates withmultiple copies of the HDV-encoded proteins to assemble aribonucleoprotein (RNP) complex. The RNP is exported from the cell bythe HBV envelope proteins, which are able to assemble lipoproteinvesicles that bud into the lumen of a pre-Golgi compartment before beingsecreted. Moreover, the HBV envelope proteins also provide a mechanismfor the targeting of HDV to an uninfected cell, thereby ensuring thespread of HDV.

The complications caused by HDV include a greater likelihood ofexperiencing liver failure in acute infections and a rapid progressionto liver cirrhosis, with an increased chance of developing liver cancerin chronic infections. In combination with hepatitis B virus, hepatitisD has the highest fatality rate of all the hepatitis infections, at 20%(Fattovich G, Giustina G, Christensen E, Pantalena M, Zagni I, Realdi G,Schalm S W. Influence of hepatitis delta virus infection on morbidityand mortality in compensated cirrhosis type B. Gut. 2000 March;46(3):420-6). The only approved therapy for chronic HDV infection isinterferon-alpha. However, treatment of HDV with interferon-alpha isrelatively inefficient and not well-tolerated. Treatment withinterferon-alpha results in sustained virological response six monthspost-treatment in one fourth of the patients. Also, nucleos(t)ideanalogs (NAs) have been widely tested in hepatitis delta, but theyappear to be ineffective. Combination treatment of NAs with interferonalso proved to be disappointing and so there is a need for noveltherapeutic options (Zaigham Abbas, Minaam Abbas Management of hepatitisdelta: Need for novel therapeutic Options. World J Gastroenterol 2015Aug. 28; 21(32): 9461-9465).

In view of the above, it is the object of the present invention providean antibody-based product, which is capable of neutralizing both,hepatitis B virus (HBV) and hepatitis delta virus (HDV). That enables animproved prevention and treatment of hepatitis B. Moreover, no treatmentis presently available for hepatitis D and, thus, it is also an objectof the present invention to provide an antibody-based product forprevention and treatment of hepatitis D. It is furthermore an object ofthe present invention to provide an antibody-based product, whichenables a better treatment of chronic hepatitis B. To this end, it isadvantageous if one single antibody-based product acts in different waysby (i) potently neutralizing HBV, (ii) promoting clearance of HBsAg andHBV and (iii) inducing seroconversion, i.e. an immune response to thevirus. Moreover, antibodies may advantageously also promote an improvedpresentation of the antigen, thereby facilitating the restoration of ananti-HBV T-cell response.

In addition, it is an object of the present invention to provide anantibody, or an antigen-binding fragment thereof, which binds todifferent—preferably all known—genotypes of hepatitis B virus surfaceantigen and to different—preferably all known—infectious mutants ofhepatitis B virus surface antigen. In summary, it is the object of thepresent invention to provide improved antibodies, or antigen bindingfragments thereof, as well as related nucleic acid molecules, vectorsand cells and pharmaceutical compositions, which overcome the abovediscussed drawbacks of the prior art.

The object underlying the present invention is solved by thesubject-matter set out below and in the appended claims.

In the following, the elements of the present invention will bedescribed. These elements are listed with specific embodiments, however,it should be understood that they may be combined in any manner and inany number to create additional embodiments. This description should beunderstood to support and encompass embodiments which combine theexplicitly described embodiments with any number of the disclosed and/orpreferred elements. Furthermore, any permutations and combinations ofall described elements in this application should be considereddisclosed by the description of the present application unless thecontext indicates otherwise.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art.

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the term “comprise”, and variations such as“comprises” and “comprising”, will be understood to imply the inclusionof a stated member, integer or step but not the exclusion of any othernon-stated member, integer or step. The term “consist of” is aparticular embodiment of the term “comprise”, wherein any othernon-stated member, integer or step is excluded. In the context of thepresent invention, the term “comprise” encompasses the term “consistof”. The term “comprising” thus encompasses “including” as well as“consisting” e.g., a composition “comprising” X may consist exclusivelyof X or may include something additional e.g., X+Y.

The terms “a” and “an” and “the” and similar reference used in thecontext of describing the invention (especially in the context of theclaims) are to be construed to cover both the singular and the plural,unless otherwise indicated herein or clearly contradicted by context.Recitation of ranges of values herein is merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range. Unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein. No language in the specification should beconstrued as indicating any non-claimed element essential to thepractice of the invention.

The word “substantially” does not exclude “completely” e.g., acomposition which is “substantially free” from Y may be completely freefrom Y. Where necessary, the word “substantially” may be omitted fromthe definition of the invention.

The term “about” in relation to a numerical value x means x±10%.

The term “disease” as used herein is intended to be generallysynonymous, and is used interchangeably with, the terms “disorder” and“condition” (as in medical condition), in that all reflect an abnormalcondition of the human or animal body or of one of its parts thatimpairs normal functioning, is typically manifested by distinguishingsigns and symptoms, and causes the human or animal to have a reducedduration or quality of life.

As used herein, reference to “treatment” of a subject or patient isintended to include prevention, prophylaxis, attenuation, ameliorationand therapy. The terms “subject” or “patient” are used interchangeablyherein to mean all mammals including humans. Examples of subjectsinclude humans, cows, dogs, cats, horses, goats, sheep, pigs, andrabbits. In one embodiment, the patient is a human.

As used herein, the terms “peptide”, “polypeptide”, and “protein” andvariations of these terms refer to a molecule, in particular a peptide,oligopeptide, polypeptide or protein including fusion protein,respectively, comprising at least two amino acids joined to each otherby a normal peptide bond, or by a modified peptide bond, such as forexample in the cases of isosteric peptides. For example, a peptide,polypeptide or protein is preferably composed of amino acids selectedfrom the 20 amino acids defined by the genetic code, linked to eachother by a normal peptide bond (“classical” polypeptide). A peptide,polypeptide or protein can be composed of L-amino acids and/or D-aminoacids. In particular, the terms “peptide”, “polypeptide”, “protein” alsoinclude “peptidomimetics” which are defined as peptide analogscontaining non-peptidic structural elements, which peptides are capableof mimicking or antagonizing the biological action(s) of a naturalparent peptide. A peptidomimetic lacks classical peptide characteristicssuch as enzymatically scissile peptide bonds. In particular, a peptide,polypeptide or protein may comprise amino acids other than the 20 aminoacids defined by the genetic code in addition to these amino acids, orit can be composed of amino acids other than the 20 amino acids definedby the genetic code. In particular, a peptide, polypeptide or protein inthe context of the present invention can equally be composed of aminoacids modified by natural processes, such as post-translationalmaturation processes or by chemical processes, which are well known to aperson skilled in the art. Such modifications are fully detailed in theliterature. These modifications can appear anywhere in the polypeptide:in the peptide skeleton, in the amino acid chain or even at the carboxy-or amino-terminal ends. In particular, a peptide or polypeptide can bebranched following an ubiquitination or be cyclic with or withoutbranching. This type of modification can be the result of natural orsynthetic post-translational processes that are well known to a personskilled in the art. The terms “peptide”, “polypeptide”, “protein” in thecontext of the present invention in particular also include modifiedpeptides, polypeptides and proteins. For example, peptide, polypeptideor protein modifications can include acetylation, acylation,ADP-ribosylation, amidation, covalent fixation of a nucleotide or of anucleotide derivative, covalent fixation of a lipid or of a lipidicderivative, the covalent fixation of a phosphatidylinositol, covalent ornon-covalent cross-linking, cyclization, disulfide bond formation,demethylation, glycosylation including pegylation, hydroxylation,iodization, methylation, myristoylation, oxidation, proteolyticprocesses, phosphorylation, prenylation, racemization, seneloylation,sulfatation, amino acid addition such as arginylation or ubiquitination.Such modifications are fully detailed in the literature (ProteinsStructure and Molecular Properties (1993) 2nd Ed., T. E. Creighton, NewYork; Post-translational Covalent Modifications of Proteins (1983) B. C.Johnson, Ed., Academic Press, New York; Seifter et al. (1990) Analysisfor protein modifications and nonprotein cofactors, Meth. Enzymol. 182:626-646 and Rattan et al., (1992) Protein Synthesis: Post-translationalModifications and Aging, Ann NY Acad Sci, 663: 48-62). Accordingly, theterms “peptide”, “polypeptide”, “protein” preferably include for examplelipopeptides, lipoproteins, glycopeptides, glycoproteins and the like.

As used herein a “(poly)peptide” comprises a single chain of amino acidmonomers linked by peptide bonds as explained above. A “protein”, asused herein, comprises one or more, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10(poly)peptides, i.e. one or more chains of amino acid monomers linked bypeptide bonds as explained above. Preferably, a protein according to thepresent invention comprises 1, 2, 3, or 4 polypeptides.

The term “recombinant”, as used herein (e.g. a recombinant antibody, arecombinant protein, a recombinant nucleic acid etc.), refers to anymolecule (antibody, protein, nucleic acid etc.) which is prepared,expressed, created or isolated by recombinant means, and which is notnaturally occurring.

As used herein, the terms “nucleic acid”, “nucleic acid molecule” and“polynucleotide” are used interchangeably and are intended to includeDNA molecules and RNA molecules. A nucleic acid molecule may besingle-stranded or double-stranded, but preferably is double-strandedDNA.

As used herein, the terms “cell,” “cell line,” and “cell culture” areused interchangeably and all such designations include progeny. Thus,the words “transformants” and “transformed cells” include the primarysubject cell and cultures derived therefrom without regard for thenumber of transfers. It is also understood that all progeny may not beprecisely identical in DNA content, due to deliberate or inadvertentmutations. Variant progeny that have the same function or biologicalactivity as screened for in the originally transformed cell areincluded. Where distinct designations are intended, it will be clearfrom the context.

As used herein, the terms “antigen binding fragment,” “fragment,” and“antibody fragment” are used interchangeably to refer to any fragment ofan antibody of the invention that retains the antigen-binding activityof the antibody. Examples of antibody fragments include, but are notlimited to, a single chain antibody, Fab, Fab′, F(ab′)₂, Fv or scFv.Further, the term “antibody” as used herein includes both antibodies andantigen binding fragments thereof.

As used herein, a “neutralizing antibody” is one that can neutralize,i.e., prevent, inhibit, reduce, impede or interfere with, the ability ofa pathogen to initiate and/or perpetuate an infection in a host. Theterms “neutralizing antibody” and “an antibody that neutralizes” or“antibodies that neutralize” are used interchangeably herein. Theseantibodies can be used alone, or in combination, as prophylactic ortherapeutic agents upon appropriate formulation, in association withactive vaccination, as a diagnostic tool, or as a production tool asdescribed herein.

Doses are often expressed in relation to the bodyweight. Thus, a dosewhich is expressed as [g, mg, or other unit]/kg (or g, mg etc.) usuallyrefers to [g, mg, or other unit] “per kg (or g, mg etc.) bodyweight”,even if the term “bodyweight” is not explicitly mentioned.

The terms “binding” and “specifically binding” and similar reference donot encompass non-specific sticking.

The term “vaccine” as used herein is typically understood to be aprophylactic or therapeutic material providing at least one antigen,preferably an immunogen. The antigen or immunogen may be derived fromany material that is suitable for vaccination. For example, the antigenor immunogen may be derived from a pathogen, such as from bacteria orvirus particles etc., or from a tumor or cancerous tissue. The antigenor immunogen stimulates the body's adaptive immune system to provide anadaptive immune response. In particular, an “antigen” or an “immunogen”refers typically to a substance which may be recognized by the immunesystem, preferably by the adaptive immune system, and which is capableof triggering an antigen-specific immune response, e.g. by formation ofantibodies and/or antigen-specific T cells as part of an adaptive immuneresponse. Typically, an antigen may be or may comprise a peptide orprotein which may be presented by the MHC to T-cells.

As used herein, the term “sequence variant” refers to any sequencehaving one or more alterations in comparison to a reference sequence,whereby a reference sequence is any of the sequences listed in the“Table of Sequences and SEQ ID Numbers” (sequence listing), i.e. SEQ IDNO: 1 to SEQ ID NO: 88. Thus, the term “sequence variant” includesnucleotide sequence variants and amino acid sequence variants. For asequence variant in the context of a nucleotide sequence, the referencesequence is also a nucleotide sequence, whereas for sequence variant inthe context of an amino acid sequence, the reference sequence is also anamino acid sequence. A “sequence variant” as used herein is at least80%, preferably at least 85%, more preferably at least 90%, even morepreferably at least 95% and particularly preferably at least 98% or 99%identical to the reference sequence. Sequence identity is usuallycalculated with regard to the full length of the reference sequence(i.e. the sequence recited in the application). Percentage identity, asreferred to herein, can be determined, for example, using BLAST usingthe default parameters specified by the NCBI (the National Center forBiotechnology Information; http://www.ncbi.nlm.nih.gov/) [Blosum 62matrix; gap open penalty=11 and gap extension penalty=1].

A “sequence variant” in the context of a nucleic acid (nucleotide)sequence has an altered sequence in which one or more of the nucleotidesin the reference sequence is deleted, or substituted, or one or morenucleotides are inserted into the sequence of the reference nucleotidesequence. Nucleotides are referred to herein by the standard one-letterdesignation (A, C, G, or T). Due to the degeneracy of the genetic code,a “sequence variant” of a nucleotide sequence can either result in achange in the respective reference amino acid sequence, i.e. in an aminoacid “sequence variant” or not. Nucleotide sequence variants, which donot result in amino acid sequence variants are preferred (silentmutations). However, nucleotide sequence variants leading to“non-silent” mutations are also within the scope, in particular suchnucleotide sequence variants, which result in an amino acid sequence,which is at least 80%, preferably at least 85%, more preferably at least90%, even more preferably at least 95% and particularly preferably atleast 98% or 99% identical to the reference amino acid sequence.

A “sequence variant” in the context of an amino acid sequence has analtered sequence in which one or more of the amino acids is deleted,substituted or inserted in comparison to the reference amino acidsequence. As a result of the alterations, such a sequence variant has anamino acid sequence which is at least 80%, preferably at least 85%, morepreferably at least 90%, even more preferably at least 95% andparticularly preferably at least 98% or 99% identical to the referenceamino acid sequence. For example, per 100 amino acids of the referencesequence a variant sequence has no more than 10 alterations, i.e. anycombination of deletions, insertions or substitutions, is “at least 90%identical” to the reference sequence.

While it is possible to have non-conservative amino acid substitutions,it is preferred that the substitutions be conservative amino acidsubstitutions, in which the substituted amino acid has similarstructural or chemical properties with the corresponding amino acid inthe reference sequence. By way of example, conservative amino acidsubstitutions involve substitution of one aliphatic or hydrophobic aminoacids, e.g. alanine, valine, leucine and isoleucine, with another;substitution of one hydoxyl-containing amino acid, e.g. serine andthreonine, with another; substitution of one acidic residue, e.g.glutamic acid or aspartic acid, with another; replacement of oneamide-containing residue, e.g. asparagine and glutamine, with another;replacement of one aromatic residue, e.g. phenylalanine and tyrosine,with another; replacement of one basic residue, e.g. lysine, arginineand histidine, with another; and replacement of one small amino acid,e.g., alanine, serine, threonine, methionine, and glycine, with another.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includethe fusion to the N- or C-terminus of an amino acid sequence to areporter molecule or an enzyme.

Importantly, the alterations in the sequence variants do not abolish thefunctionality of the respective reference sequence, in the present case,for example the functionality of a sequence of an antibody, or antigenbinding fragment thereof, to bind to the same epitope and/or tosufficiently neutralize infection of HBV and HDV. Guidance indetermining which nucleotides and amino acid residues, respectively, maybe substituted, inserted or deleted without abolishing suchfunctionality can be found by using computer programs well known in theart.

As used herein, a nucleic acid sequence or an amino acid sequence“derived from” a designated nucleic acid, peptide, polypeptide orprotein refers to the origin of the nucleic acid, peptide, polypeptideor protein. Preferably, the nucleic acid sequence or amino acid sequencewhich is derived from a particular sequence has an amino acid sequencethat is essentially identical to that sequence or a portion thereof,from which it is derived, whereby “essentially identical” includessequence variants as defined above. Preferably, the nucleic acidsequence or amino acid sequence which is derived from a particularpeptide or protein, is derived from the corresponding domain in theparticular peptide or protein. Thereby, “corresponding” refers inparticular to the same functionality. For example, an “extracellulardomain” corresponds to another “extracellular domain” (of anotherprotein), or a “transmembrane domain” corresponds to another“transmembrane domain” (of another protein). “Corresponding” parts ofpeptides, proteins and nucleic acids are thus easily identifiable to oneof ordinary skill in the art. Likewise, sequences “derived from” othersequence are usually easily identifiable to one of ordinary skill in theart as having its origin in the sequence.

Preferably, a nucleic acid sequence or an amino acid sequence derivedfrom another nucleic acid, peptide, polypeptide or protein may beidentical to the starting nucleic acid, peptide, polypeptide or protein(from which it is derived). However, a nucleic acid sequence or an aminoacid sequence derived from another nucleic acid, peptide, polypeptide orprotein may also have one or more mutations relative to the startingnucleic acid, peptide, polypeptide or protein (from which it isderived), in particular a nucleic acid sequence or an amino acidsequence derived from another nucleic acid, peptide, polypeptide orprotein may be a functional sequence variant as described above of thestarting nucleic acid, peptide, polypeptide or protein (from which it isderived). For example, in a peptide/protein one or more amino acidresidues may be substituted with other amino acid residues or one ormore amino acid residue insertions or deletions may occur.

As used herein, the term “mutation” relates to a change in the nucleicacid sequence and/or in the amino acid sequence in comparison to areference sequence, e.g. a corresponding genomic sequence. A mutation,e.g. in comparison to a genomic sequence, may be, for example, a(naturally occurring) somatic mutation, a spontaneous mutation, aninduced mutation, e.g. induced by enzymes, chemicals or radiation, or amutation obtained by site-directed mutagenesis (molecular biologymethods for making specific and intentional changes in the nucleic acidsequence and/or in the amino acid sequence). Thus, the terms “mutation”or “mutating” shall be understood to also include physically making amutation, e.g. in a nucleic acid sequence or in an amino acid sequence.A mutation includes substitution, deletion and insertion of one or morenucleotides or amino acids as well as inversion of several successivenucleotides or amino acids.

To achieve a mutation in an amino acid sequence, preferably a mutationmay be introduced into the nucleotide sequence encoding said amino acidsequence in order to express a (recombinant) mutated polypeptide. Amutation may be achieved e.g., by altering, e.g., by site-directedmutagenesis, a codon of a nucleic acid molecule encoding one amino acidto result in a codon encoding a different amino acid, or by synthesizinga sequence variant, e.g., by knowing the nucleotide sequence of anucleic acid molecule encoding a polypeptide and by designing thesynthesis of a nucleic acid molecule comprising a nucleotide sequenceencoding a variant of the polypeptide without the need for mutating oneor more nucleotides of a nucleic acid molecule.

The present invention is based, amongst other findings, on the discoveryand isolation of antibodies that are highly potent in neutralizinghepatitis B and hepatitis delta viruses, as well as of epitopes to whichthe antibodies of the invention bind. Such antibodies are desirable, asonly small quantities of the antibodies are required in order toneutralize hepatitis B virus. Moreover, there is currently no treatmentavailable for hepatitis D. The antibodies according to the presentinvention are highly effective in preventing as well as treating orattenuating HBV and HDV. Moreover, the antibodies according to thepresent invention bind to different—preferably all known—genotypes ofhepatitis B virus surface antigen and to different—preferably allknown—infectious mutants of hepatitis B virus surface antigen.

Antibodies and Antigen-Binding Fragments Thereof.

In a first aspect the present invention provides an isolated antibody,or an antigen binding fragment thereof, that binds to the antigenic loopregion of HBsAg and neutralizes infection with hepatitis B virus andhepatitis delta virus.

As used herein, the term “antibody” encompasses various forms ofantibodies including, without being limited to, whole antibodies,antibody fragments, in particular antigen binding fragments, humanantibodies, chimeric antibodies, humanized antibodies, recombinantantibodies and genetically engineered antibodies (variant or mutantantibodies) as long as the characteristic properties according to theinvention are retained. Human antibodies and monoclonal antibodies arepreferred and especially preferred are human monoclonal antibodies, inparticular as recombinant human monoclonal antibodies.

Human antibodies are well-known in the state of the art (van Dijk, M.A., and van de Winkel, J. G., Curr. Opin. Chem. Biol. 5 (2001) 368-374).Human antibodies can also be produced in transgenic animals (e.g., mice)that are capable, upon immunization, of producing a full repertoire or aselection of human antibodies in the absence of endogenousimmunoglobulin production. Transfer of the human germ-lineimmunoglobulin gene array in such germ-line mutant mice will result inthe production of human antibodies upon antigen challenge (see, e.g.,Jakobovits, A., et al., Proc. Natl. Acad. Sci. USA 90 (1993) 2551-2555;Jakobovits, A., et al., Nature 362 (1993) 255-258; Bruggemann, M., etal., Year Immunol. 7 (1993) 3340). Human antibodies can also be producedin phage display libraries (Hoogenboom, H. R., and Winter, G., J. Mol.Biol. 227 (1992) 381-388; Marks, J. D., et al., J. Mol. Biol. 222 (1991)581-597). The techniques of Cole et al. and Boerner et al. are alsoavailable for the preparation of human monoclonal antibodies (Cole etal., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss , p. 77(1985); and Boerner, P., et al., J. Immunol. 147 (1991) 86-95).Preferably, human monoclonal antibodies are prepared by using improvedEBV-B cell immortalization as described in Traggiai E, Becker S,Subbarao K, Kolesnikova L, Uematsu Y, Gismondo M R, Murphy B R, RappuoliR, Lanzavecchia A. (2004): An efficient method to make human monoclonalantibodies from memory B cells: potent neutralization of SARScoronavirus. Nat Med. 10(8):871-5. The term “human antibody” as usedherein also comprises such antibodies which are modified, e.g. in thevariable region, to generate the properties according to the inventionas described herein. As used herein, the term “variable region”(variable region of a light chain (V_(L)), variable region of a heavychain (V_(H))) denotes each of the pair of light and heavy chains whichis involved directly in binding the antibody to the antigen.

Antibodies of the invention can be of any isotype (e.g., IgA, IgG, IgMi.e. an α, γ or μ heavy chain), but will preferably be IgG. Within theIgG isotype, antibodies may be IgG1, IgG2, IgG3 or IgG4 subclass,whereby IgG1 is preferred. Antibodies of the invention may have a κ or aλ light chain. HBsAg-specific antibodies of the IgG-type mayadvantageously also block the release of HBV and HBsAg from infectedcells—based on antigen-independent uptake of IgG through FcRN-IgGreceptors into hepatocytes. Therefore, HBsAg-specific antibodies of theIgG-type can bind intracellularly and thereby block the release of HBVvirions and HBsAg.

Preferably, the antibody according to the present invention, or theantigen binding fragment thereof, is a purified antibody, a single chainantibody, Fab, Fab′, F(ab′)2, Fv or scFv.

The antibodies of the invention may thus preferably be human antibodies,monoclonal antibodies, human monoclonal antibodies, recombinantantibodies or purified antibodies. The invention also provides fragmentsof the antibodies of the invention, particularly fragments that retainthe antigen-binding activity of the antibodies. Such fragments include,but are not limited to, single chain antibodies, Fab, Fab′, F(ab′)2, Fvor scFv. Although the specification, including the claims, may, in someplaces, refer explicitly to antigen binding fragment(s), antibodyfragment(s), variant(s) and/or derivative(s) of antibodies, it isunderstood that the term “antibody” or “antibody of the invention”includes all categories of antibodies, namely, antigen bindingfragment(s), antibody fragment(s), variant(s) and derivative(s) ofantibodies.

Fragments of the antibodies of the invention can be obtained from theantibodies by methods that include digestion with enzymes, such aspepsin or papain, and/or by cleavage of disulfide bonds by chemicalreduction. Alternatively, fragments of the antibodies can be obtained bycloning and expression of part of the sequences of the heavy or lightchains. Antibody “fragments” include Fab, Fab′, F(ab′)2 and Fvfragments. The invention also encompasses single-chain Fv fragments(scFv) derived from the heavy and light chains of an antibody of theinvention. For example, the invention includes a scFv comprising theCDRs from an antibody of the invention. Also included are heavy or lightchain monomers and dimers, single domain heavy chain antibodies, singledomain light chain antibodies, as well as single chain antibodies, e.g.,single chain Fv in which the heavy and light chain variable domains arejoined by a peptide linker.

Antibody fragments of the invention may impart monovalent or multivalentinteractions and be contained in a variety of structures as describedabove. For instance, scFv molecules may be synthesized to create atrivalent “triabody” or a tetravalent “tetrabody.” The scFv moleculesmay include a domain of the Fc region resulting in bivalent minibodies.In addition, the sequences of the invention may be a component ofmultispecific molecules in which the sequences of the invention targetthe epitopes of the invention and other regions of the molecule bind toother targets. Exemplary molecules include, but are not limited to,bispecific Fab2, trispecific Fabs, bispecific scFv, and diabodies(Holliger and Hudson, 2005, Nature Biotechnology 9: 1126-1136).

Antibodies according to the present invention may be provided inpurified form. Typically, the antibody will be present in a compositionthat is substantially free of other polypeptides e.g., where less than90% (by weight), usually less than 60% and more usually less than 50% ofthe composition is made up of other polypeptides.

Antibodies according to the present invention may be immunogenic inhuman and/or in non-human (or heterologous) hosts e.g., in mice. Forexample, the antibodies may have an idiotope that is immunogenic innon-human hosts, but not in a human host. Antibodies of the inventionfor human use include those that cannot be easily isolated from hostssuch as mice, goats, rabbits, rats, non-primate mammals, etc. and cannotgenerally be obtained by humanization or from xeno-mice.

The antibody, and the antigen binding fragment thereof, according to thepresent invention binds to the antigenic loop region of HBsAg. Theenvelope of the hepatitis B virus contains three “HBV envelope proteins”(also known as “HBsAg”, “hepatitis B surface antigen”): S protein (for“small”, also referred to as S-HBsAg), M protein (for “middle”, alsoreferred to as M-HBsAg) and L protein (for “large”, also referred to asL-HBsAg). S-HBsAg, M-HBsAg and L-HBsAg share the same C-terminalextremity (also referred to as “S domain”, 226 amino acids), whichcorresponds to the S protein (S-HBsAg) and which is crucial for virusassembly and infectivity. S-HBsAg, M-HBsAg and L-HBsAg are synthesizedin the endoplasmic reticulum (ER), assembled, and secreted as particlesthrough the Golgi apparatus. The S domain comprises four predictedtransmembrane (TM) domains, whereby both, the N-terminus as well as theC-terminus of the S domain are exposed to the lumen. The transmembranedomains TM1 and TM2 are both necessary for cotranslational proteinintegration into the ER membrane and the transmembrane domains TM3 andTM4 are located in the C-terminal third of the S domain. The “antigenicloop region” of HBsAg is located between the predicted TM3 and TM4transmembrane domains of the S domain of HBsAg, whereby the antigenicloop region comprises amino acids 101-172 of the S domain, whichcontains 226 amino acids in total (Salisse J. and Sureau C., 2009,Journal of Virology 83: 9321-9328). It is important to note that adeterminant of infectivity resides in the antigenic loop region of HBVenvelope proteins. In particular, residues between 119 and 125 of theHBsAg contained a CXXC motif, which had been demonstrated to be the mostimportant sequence required for the infectivity of HBV and HDV (Jaoude GA, Sureau C, Journal of Virology, 2005; 79:10460-6).

When it is herein referred to positions in the amino acid sequence ofthe S domain of HBsAg, it is referred to an amino acid sequence as setforth in SEQ ID NO: 3 (shown below) or to natural or artificial sequencevariants thereof.

MENITSGFLGPLLVLQAGFFLLTRILTIPQSLDSWWTSLNFLGGTTVCLGQNSQSPTSNHSPTSCPPTCPGYRWMCLRRFIIFLFILLLCLIFLLVLLDYQGMLPVCPLIPGSSTTSTGPCRTCMTTAQGTSMYPSCCCTKPSDGNCTCIPIPSSWAFGKFLWEWASARFSWLSLLVPFVQWFVGLSPTVWLSVIWMMWYWGPSLYSILSPFLPLLPIFFCLWVYI (SEQ ID NO: 3; amino acids 101-172 are shownunderlined)

For example, the expression “amino acids 101-172 of the S domain” refersto the amino acid residues from positions 101-172 of the polypeptideaccording to SEQ ID NO: 3. However, a person skilled in the artunderstands that mutations or variations (including, but not limited to,substitution, deletion and/or addition, for example, HBsAg of adifferent genotype or a different HBsAg mutant as described herein) mayoccur naturally in the amino acid sequence of the S domain of HBsAg orbe introduced artificially into the amino acid sequence of the S domainof HBsAg without affecting its biological properties. Therefore, in theinvention, the term “S domain of HBsAg” intends to comprise all suchpolypeptides, for example, including the polypeptide according to SEQ IDNO: 3 and its natural or artificial mutants. In addition, when sequencefragments of the S domain of HBsAg are described herein (e.g. aminoacids 101-172 or amino acids 120-130 of the S domain of HBsAg), theyinclude not only the corresponding sequence fragments of SEQ ID NO: 3,but also the corresponding sequence fragments of its natural orartificial mutants. For example, the expression “amino acid residuesfrom positions 101-172 of the S domain of HBsAg” comprises amino acidresidues from positions 101-172 of SEQ ID NO: 3 and the correspondingfragments of its mutants (natural or artificial mutants). According tothe invention, the expression “corresponding sequence fragments” or“corresponding fragments” refers to fragments that are located in equalpositions of sequences when the sequences are subjected to optimizedalignment, namely, the sequences are aligned to obtain a highestpercentage of identity.

The M protein (M-HBsAg) corresponds to the S protein extended by anN-terminal domain of 55 amino acids called “pre-S2”. The L protein(L-HBsAg) corresponds to the M protein extended by an N-terminal domainof 108 amino acids called “pre-S1” (genotype D). The pre-S1 and pre-S2domains of the L protein can be present either at the inner face ofviral particles (on the cytoplasmic side of the ER), playing a crucialrole in virus assembly, or on the outer face (on the luminal side of theER), available for the interaction with target cells and necessary forviral infectivity. Moreover, HBV surface proteins (HBsAgs) are not onlyincorporated into virion envelopes but also spontaneously bud fromER-Golgi intermediate compartment membranes to form empty “subviralparticles” (SVPs) that are released from the cell by secretion.

Since all three HBV envelope proteins S-HBsAg, M-HBsAg and L-HBsAgcomprise the S domain, all three HBV envelope proteins S-HBsAg, M-HBsAgand L-HBsAg also comprise the “antigenic loop region”. Accordingly, anantibody, or an antigen binding fragment thereof, according to thepresent invention, which neutralizes HBV and binds to the antigenic loopregion of HBsAg, binds to all three HBV envelope proteins S-HBsAg,M-HBsAg and L-HBsAg.

Moreover, an antibody, or an antigen binding fragment thereof, accordingto the present invention, neutralizes infection with hepatitis B virusand hepatitis delta virus. In other words, the antibody, or the antigenbinding fragment thereof, according to the present invention, reducesviral infectivity of hepatitis B virus and hepatitis delta virus.

To study and quantitate virus infectivity (or “neutralization”) in thelaboratory the person skilled in the art knows various standard“neutralization assays”. For a neutralization assay animal viruses aretypically propagated in cells and/or cell lines. In the context of thepresent invention a neutralization assay is preferred, wherein culturedcells are incubated with a fixed amount of HBV or HDV in the presence(or absence) of the antibody to be tested. As a readout the levels ofhepatitis B surface antigen (HBsAg) or hepatitis B e antigen (HBeAg)secreted into the cell culture supernatant may be used and/or HBcAgstaining may be assessed. For HDV, for example delta antigenimmunofluorescence staining may be assessed.

In a preferred embodiment of a HBV neutralization assay, cultured cells,for example HepaRG cells, in particular differentiated HepaRG cells, areincubated with a fixed amount of HBV in the presence or absence of theantibody to be tested, for example for 16 hours at 37° C. Thatincubation is preferably performed in a medium (e.g. supplemented with4% PEG 8000). After incubation, cells may be washed and furthercultivated. To measure virus infectivity, the levels of hepatitis Bsurface antigen (HBsAg) and hepatitis B e antigen (HBeAg) secreted intothe culture supernatant, e.g. from day 7 to day 11 post-infection, maybe determined by enzyme-linked immunosorbent assay (ELISA).Additionally, HBcAg staining may be assessed in an immunofluorescenceassay. In a preferred embodiment of a HDV neutralization assayessentially the same assay as for HBV may be used, with the differencethat sera from HDV carriers may be used as HDV infection inoculum ondifferentiated HepaRg cells (instead of HBV). For detection, deltaantigen immunofluorescence staining may be used as a readout.

The antibody and antigen binding fragment of the invention have highneutralizing potency. The concentration of the antibody of the inventionrequired for 50% neutralization of hepatitis B virus (HBV) and hepatitisdelta virus (HDV), is, for example, about 10 μg/ml or less. Preferably,the concentration of the antibody of the invention required for 50%neutralization of HBV and HDV is about 5 μg/ml, more preferably theconcentration of the antibody of the invention required for 50%neutralization of HBV and HDV is about 1 g/ml, even more preferably, theconcentration of the antibody of the invention required for 50%neutralization of HBV and HDV is about 750 ng/ml. Most preferably, theconcentration of the antibody of the invention required for 50%neutralization of HBV and HDV is 500 ng/ml or less, e.g. 450, 400, 350,300, 250, 200, 175, 150, 125, 100, 90, 80, 70, 60 or about 50 ng/ml orless. This means that only low concentrations of the antibody arerequired for 50% neutralization of HBV and HDV. Specificity and potencycan be measured using standard assays as known to one of skill in theart.

The antibody, or the antigen binding fragment thereof, according to thepresent invention, which potently neutralizes both, HBV and HDV, isuseful in the prevention and treatment of hepatitis B and hepatitis D.In this context it is of note that infection with HDV typically occurssimultaneously or subsequently to infection with HBV (inoculation withHDV in the absence of HBV does not cause hepatitis D since HDV requiresthe support of HBV for its own replication) and hepatitis D is typicallyobserved in chronic HBV carriers.

Preferably, the antibody according to the present invention, or anantigen binding fragment thereof, promotes clearance of HBsAg and HBV.In particular, the antibody according to the present invention, or anantigen binding fragment thereof, promotes clearance of both, HBV andsubviral particles of hepatitis B virus (SVP's). Clearance of HBsAg orof subviral particles may be assessed by measuring the level of HBsAgfor example in a blood sample, e.g. from a hepatitis B patient.Similarly, clearance of HBV may be assessed by measuring the level ofHBV for example in a blood sample, e.g. from a hepatitis B patient.

In the sera of patients infected with HBV, in addition to infectiousparticles (HBV), there is typically an excess (typically 1,000- to100,000-fold) of empty subviral particles (SVP) composed solely of HBVenvelope proteins (HBsAg) in the form of relatively smaller spheres andfilaments of variable length. Subviral particles were shown to stronglyenhance intracellular viral replication and gene expression of HBV(Bruns M. et al. 1998 J Virol 72(2): 1462-1468). This is also importantin the context of infectivity of sera containing HBV, since theinfectivity depends not only on the number of viruses but also on thenumber of SVP's (Bruns M. et al. 1998 J Virol 72(2): 1462-1468).Moreover, an excess of subviral particles can serve as a decoy byabsorbing neutralizing antibodies and therefore delay the clearance ofinfection. Typically, achievement of hepatitis B surface antigen (HBsAg)loss is thus considered to be an ideal endpoint of treatment and theclosest outcome to cure chronic hepatitis B (CHB).

Accordingly, the antibody according to the present invention, or anantigen binding fragment thereof, which preferably promotes clearance ofHBsAg, in particular clearance of subviral particles of hepatitis Bvirus, and HBV enables improved treatment of hepatitis B, in particularin the context of chronic hepatitis B. Thereby, the antibody accordingto the present invention, or an antigen binding fragment thereof, canexert its neutralization properties even more potent since less of theantibody is absorbed by SVP's acting as a decoy. In addition, theantibody according to the present invention, or an antigen bindingfragment thereof, which preferably promotes clearance of subviralparticles of hepatitis B virus, decreases infectivity of sera containingHBV.

Preferably, the antibody according to the present invention, or anantigen binding fragment thereof, comprises an Fc moiety. Morepreferably, the Fc moiety is derived from human origin, e.g. from humanIgG1, IgG2, IgG3, and/or IgG4, whereby human IgG1 is particularlypreferred.

As used herein, the term “Fc moiety” refers to a sequence derived fromthe portion of an immunoglobulin heavy chain beginning in the hingeregion just upstream of the papain cleavage site (e.g., residue 216 innative IgG, taking the first residue of heavy chain constant region tobe 114) and ending at the C-terminus of the immunoglobulin heavy chain.Accordingly, an Fc moiety may be a complete Fc moiety or a portion(e.g., a domain) thereof. A complete Fc moiety comprises at least ahinge domain, a CH2 domain, and a CH3 domain (e.g., EU amino acidpositions 216-446). An additional lysine residue (K) is sometimespresent at the extreme C-terminus of the Fc moiety, but is often cleavedfrom a mature antibody. Each of the amino acid positions within an Fcmoiety have been numbered according to the art-recognized EU numberingsystem of Kabat, see e.g., by Kabat et al., in “Sequences of Proteins ofImmunological Interest”, U.S. Dept. Health and Human Services, 1983 and1987.

Preferably, in the context of the present invention an Fc moietycomprises at least one of: a hinge (e.g., upper, middle, and/or lowerhinge region) domain, a CH2 domain, a CH3 domain, or a variant, portion,or fragment thereof. In preferred embodiments, an Fc moiety comprises atleast a hinge domain, a CH2 domain or a CH3 domain. More preferably, theFc moiety is a complete Fc moiety. The Fc moiety may also comprises oneor more amino acid insertions, deletions, or substitutions relative to anaturally-occurring Fc moiety. For example, at least one of a hingedomain, CH2 domain or CH3 domain (or portion thereof) may be deleted.For example, an Fc moiety may comprise or consist of: (i) hinge domain(or portion thereof) fused to a CH2 domain (or portion thereof), (ii) ahinge domain (or portion thereof) fused to a CH3 domain (or portionthereof), (iii) a CH2 domain (or portion thereof) fused to a CH3 domain(or portion thereof), (iv) a hinge domain (or portion thereof), (v) aCH2 domain (or portion thereof), or (vi) a CH3 domain or portionthereof.

It will be understood by one of ordinary skill in the art that the Fcmoiety may be modified such that it varies in amino acid sequence fromthe complete Fc moiety of a naturally occurring immunoglobulin molecule,while retaining at least one desirable function conferred by thenaturally-occurring Fc moiety. Such functions include Fc receptor (FcR)binding, antibody half-life modulation, ADCC function, protein Abinding, protein G binding, and complement binding. The portions ofnaturally occurring Fc moieties, which are responsible and/or essentialfor such functions are well known by those skilled in the art.

For example, to activate the complement cascade C1q binds to at leasttwo molecules of IgG1 or one molecule of IgM, attached to the antigenictarget (Ward, E. S., and Ghetie, V., Ther. Immunol. 2 (1995) 77-94).Burton, D. R., described (Mol. Immunol. 22 (1985) 161-206) that theheavy chain region comprising amino acid residues 318 to 337 is involvedin complement fixation. Duncan, A. R., and Winter, G. (Nature 332 (1988)738-740), using site directed mutagenesis, reported that Glu318, Lys320and Lys322 form the binding site to C1q. The role of Glu318, Lys320 andLys 322 residues in the binding of C1q was confirmed by the ability of ashort synthetic peptide containing these residues to inhibit complementmediated lysis.

For example, FcR binding can be mediated by the interaction of the Fcmoiety (of an antibody) with Fc receptors (FcRs), which are specializedcell surface receptors on hematopoietic cells. Fc receptors belong tothe immunoglobulin superfamily, and were shown to mediate both theremoval of antibody-coated pathogens by phagocytosis of immunecomplexes, and the lysis of erythrocytes and various other cellulartargets (e.g. tumor cells) coated with the corresponding antibody, viaantibody dependent cell mediated cytotoxicity (ADCC; Van de Winkel, J.G., and Anderson, C. L., J. Leukoc. Biol. 49 (1991) 511-524). FcRs aredefined by their specificity for immunoglobulin classes; Fc receptorsfor IgG antibodies are referred to as FcγR, for IgE as FcεR, for IgA asFcαR and so on and neonatal Fc receptors are referred to as FcRn. Fcreceptor binding is described for example in Ravetch, J. V., and Kinet,J. P., Annu. Rev. Immunol. 9 (1991) 457-492; Capel, P. J., et al.,Immunomethods 4 (1994) 25-34; de Haas, M., et al., J Lab. Clin. Med. 126(1995) 330-341; and Gessner, J. E., et al., Ann. Hematol. 76 (1998)231-248.

Cross-linking of receptors by the Fc domain of native IgG antibodies(FcγR) triggers a wide variety of effector functions includingphagocytosis, antibody-dependent cellular cytotoxicity, and release ofinflammatory mediators, as well as immune complex clearance andregulation of antibody production. Therefore, Fc moieties providingcross-linking of receptors (FcγR) are preferred. In humans, threeclasses of FcγR have been characterized, which are: (i) FcγRI (CD64),which binds monomeric IgG with high affinity and is expressed onmacrophages, monocytes, neutrophils and eosinophils; (ii) FcγRII (CD32),which binds complexed IgG with medium to low affinity, is widelyexpressed, in particular on leukocytes, is known to be a central playerin antibody-mediated immunity, and which can be divided into FcγRIIA,FcγRIIB and FcγRIIC, which perform different functions in the immunesystem, but bind with similar low affinity to the IgG-Fc, and theectodomains of these receptors are highly homologuous; and (iii) FcγRIII(CD16), which binds IgG with medium to low affinity and exists as twotypes: FcγRIIIA found on NK cells, macrophages, eosinophils and somemonocytes and T cells and mediating ADCC and FcγRIIIB, which is highlyexpressed on neutrophils. FcγRIIA is found on many cells involved inkilling (e.g. macrophages, monocytes, neutrophils) and seems able toactivate the killing process. FcγRIIB seems to play a role in inhibitoryprocesses and is found on B-cells, macrophages and on mast cells andeosinophils. Importantly, 75% of all FcγRIIB is found in the liver(Ganesan, L. P. et al., 2012: FcγRIIb on liver sinusoidal endotheliumclears small immune complexes. Journal of Immunology 189: 4981-4988).FcγRIIB is abundantly expressed on Liver Sinusoidal Endothelium, calledLSEC, and in Kupffer cells in the liver and LSEC are the major site ofsmall immune complexes clearance (Ganesan, L. P. et al., 2012: FcγRIIbon liver sinusoidal endothelium clears small immune complexes. Journalof Immunology 189: 4981-4988).

Accordingly, in the present invention such antibodies, and antigenbinding fragments thereof, are preferred, which are able to bind toFcγRIIb, for example antibodies comprising an Fc moiety for binding toFcγRIIb, in particular an Fc region, such as, for example IgG-typeantibodies. Moreover, it is possible to engineer the Fc moiety toenhance FcγRIIB binding by introducing the mutations S267E and L328F asdescribed by Chu, S. Y. et al., 2008: Inhibition of B cellreceptor-mediated activation of primary human B cells by coengagement ofCD19 and FcgammaRIIb with Fc-engineered antibodies. Molecular Immunology45, 3926-3933. Thereby, the clearance of immune complexes can beenhanced (Chu, S., et al., 2014: Accelerated Clearance of IgE InChimpanzees Is Mediated By Xmab7195, An Fc-Engineered Antibody WithEnhanced Affinity For Inhibitory Receptor FcγRIIb. Am J Respir Crit,American Thoracic Society International Conference Abstracts).Accordingly, in the context of the present invention such antibodies, orantigen binding fragments thereof, are preferred, which comprise anengineered Fc moiety with the mutations S267E and L328F, in particularas described by Chu, S. Y. et al., 2008: Inhibition of B cellreceptor-mediated activation of primary human B cells by coengagement ofCD19 and FcgammaRIIb with Fc-engineered antibodies. Molecular Immunology45, 3926-3933.

On B-cells it seems to function to suppress further immunoglobulinproduction and isotype switching to say for example the IgE class. Onmacrophages, FcγRIIB acts to inhibit phagocytosis as mediated throughFcγRIIA. On eosinophils and mast cells the b form may help to suppressactivation of these cells through IgE binding to its separate receptor.

Regarding FcγRI binding, modification in native IgG of at least one ofE233-G236, P238, D265, N297, A327 and P329 reduces binding to FcγRI.IgG2 residues at positions 233-236, substituted into IgG1 and IgG4,reduces binding to FcγRI by 103-fold and eliminated the human monocyteresponse to antibody-sensitized red blood cells (Armour, K. L., et al.Eur. J. Immunol. 29 (1999) 2613-2624). Regarding FcγRII binding, reducedbinding for FcγRIIA is found e.g. for IgG mutation of at least one ofE233-G236, P238, D265, N297, A327, P329, D270, Q295, A327, R292 andK414. Regarding FcγRIII binding, reduced binding to FcγRIIIA is founde.g. for mutation of at least one of E233-G236, P238, D265, N297, A327,P329, D270, Q295, A327, S239, E269, E293, Y296, V303, A327, K338 andD376. Mapping of the binding sites on human IgG1 for Fc receptors, theabove mentioned mutation sites and methods for measuring binding toFcγRI and FcγRIIA are described in Shields, R. L., et al., J. Biol.Chem. 276 (2001) 6591-6604.

Regarding binding to the crucial FcγRII, two regions of native IgG Fcappear to be critical for interactions of FcγRIIs and IgGs, namely (i)the lower hinge site of IgG Fc, in particular amino acid residues L, L,G, G (234-237, EU numbering), and (ii) the adjacent region of the CH2domain of IgG Fc, in particular a loop and strands in the upper CH2domain adjacent to the lower hinge region, e.g. in a region of P331(Wines, B. D., et al., J. Immunol. 2000; 164: 5313-5318). Moreover,FcγRI appears to bind to the same site on IgG Fc, whereas FcRn andProtein A bind to a different site on IgG Fc, which appears to be at theCH2-CH3 interface (Wines, B. D., et al., J. Immunol. 2000; 164:5313-5318).

For example, the Fc moiety may comprise or consist of at least theportion of an Fc moiety that is known in the art to be required for FcRnbinding or extended half-life. Alternatively or additionally, the Fcmoiety of the antibody of the invention comprises at least the portionof known in the art to be required for Protein A binding and/or the Fcmoiety of the antibody of the invention comprises at least the portionof an Fc molecule known in the art to be required for protein G binding.Preferably, the retained function is the clearance of HBsAg and HBV,which is assumed to be mediated by FcγR binding. Accordingly, apreferred Fc moiety comprises at least the portion known in the art tobe required for FcγR binding. As outlined above, a preferred Fc moietymay thus at least comprise (i) the lower hinge site of native IgG Fc, inparticular amino acid residues L, L, G, G (234-237, EU numbering), and(ii) the adjacent region of the CH2 domain of native IgG Fc, inparticular a loop and strands in the upper CH2 domain adjacent to thelower hinge region, e.g. in a region of P331, for example a region of atleast 3, 4, 5, 6, 7, 8, 9, or 10 consecutive amino acids in the upperCH2 domain of native IgG Fc around P331, e.g. between amino acids 320and 340 (EU numbering) of native IgG Fc.

Preferably, the antibody, or antigen binding fragment thereof, accordingto the present invention comprises an Fc region. As used herein, theterm “Fc region” refers to the portion of an immunoglobulin formed bytwo or more Fc moieties of antibody heavy chains. For example, the Fcregion may be monomeric or “single-chain” Fc region (i.e., a scFcregion). Single chain Fc regions are comprised of Fc moieties linkedwithin a single polypeptide chain (e.g., encoded in a single contiguousnucleic acid sequence). Exemplary scFc regions are disclosed in WO2008/143954 A2. Preferably, the Fc region is a dimeric Fc region. A“dimeric Fc region” or “dcFc” refers to the dimer formed by the Fcmoieties of two separate immunoglobulin heavy chains. The dimeric Fcregion may be a homodimer of two identical Fc moieties (e.g., an Fcregion of a naturally occurring immunoglobulin) or a heterodimer of twonon-identical Fc moieties.

The Fc moieties of the Fc region may be of the same or different classand/or subclass. For example, the Fc moieties may be derived from animmunoglobulin (e.g., a human immunoglobulin) of an IgG, IgG2, IgG3 orIgG4 subclass. Preferably, the Fc moieties of Fc region are of the sameclass and subclass. However, the Fc region (or one or more Fc moietiesof an Fc region) may also be chimeric, whereby a chimeric Fc region maycomprise Fc moieties derived from different immunoglobulin classesand/or subclasses. For example, at least two of the Fc moieties of adimeric or single-chain Fc region may be from different immunoglobulinclasses and/or subclasses. Additionally or alternatively, the chimericFc regions may comprise one or more chimeric Fc moieties. For example,the chimeric Fc region or moiety may comprise one or more portionsderived from an immunoglobulin of a first subclass (e.g., an IgG1, IgG2,or IgG3 subclass) while the remainder of the Fc region or moiety is of adifferent subclass. For example, an Fc region or moiety of an Fcpolypeptide may comprise a CH2 and/or CH3 domain derived from animmunoglobulin of a first subclass (e.g., an IgG1, IgG2 or IgG4subclass) and a hinge region from an immunoglobulin of a second subclass(e.g., an IgG3 subclass). For example, the Fc region or moiety maycomprise a hinge and/or CH2 domain derived from an immunoglobulin of afirst subclass (e.g., an IgG4 subclass) and a CH3 domain from animmunoglobulin of a second subclass (e.g., an IgG1, IgG2, or IgG3subclass). For example, the chimeric Fc region may comprise an Fc moiety(e.g., a complete Fc moiety) from an immunoglobulin for a first subclass(e.g., an IgG4 subclass) and an Fc moiety from an immunoglobulin of asecond subclass (e.g., an IgG1, IgG2 or IgG3 subclass). For example, theFc region or moiety may comprise a CH2 domain from an IgG4immunoglobulin and a CH3 domain from an IgG1 immunoglobulin. Forexample, the Fc region or moiety may comprise a CH1 domain and a CH2domain from an IgG4 molecule and a CH3 domain from an IgG1 molecule. Forexample, the Fc region or moiety may comprise a portion of a CH2 domainfrom a particular subclass of antibody, e.g., EU positions 292-340 of aCH2 domain. For example, an Fc region or moiety may comprise amino acidsa positions 292-340 of CH2 derived from an IgG4 moiety and the remainderof CH2 derived from an IgG1 moiety (alternatively, 292-340 of CH2 may bederived from an IgG1 moiety and the remainder of CH2 derived from anIgG4 moiety).

Moreover, an Fc region or moiety may (additionally or alternatively) forexample comprise a chimeric hinge region. For example, the chimerichinge may be derived, e.g. in part, from an IgG1, IgG2, or IgG4 molecule(e.g., an upper and lower middle hinge sequence) and, in part, from anIgG3 molecule (e.g., an middle hinge sequence). In another example, anFc region or moiety may comprise a chimeric hinge derived, in part, froman IgG1 molecule and, in part, from an IgG4 molecule. In anotherexample, the chimeric hinge may comprise upper and lower hinge domainsfrom an IgG4 molecule and a middle hinge domain from an IgG1 molecule.Such a chimeric hinge may be made, for example, by introducing a prolinesubstitution (Ser228Pro) at EU position 228 in the middle hinge domainof an IgG4 hinge region. In another embodiment, the chimeric hinge cancomprise amino acids at EU positions 233-236 are from an IgG2 antibodyand/or the Ser228Pro mutation, wherein the remaining amino acids of thehinge are from an IgG4 antibody (e.g., a chimeric hinge of the sequenceESKYGPPCPPCPAPPVAGP). Further chimeric hinges, which may be used in theFc moiety of the antibody according to the present invention aredescribed in US 2005/0163783 A1.

In the present invention it is preferred that the Fc moiety, or the Fcregion, comprises or consists of an amino acid sequence derived from ahuman immunoglobulin sequence (e.g., from an Fc region or Fc moiety froma human IgG molecule). However, polypeptides may comprise one or moreamino acids from another mammalian species. For example, a primate Fcmoiety or a primate binding site may be included in the subjectpolypeptides. Alternatively, one or more murine amino acids may bepresent in the Fc moiety or in the Fc region.

Preferably, the antibody according to the present invention comprises,in particular in addition to an Fc moiety as described above, otherparts derived from a constant region, in particular from a constantregion of IgG, preferably from a constant region of IgG1, morepreferably from a constant region of human IgG1. More preferably, theantibody according to the present invention comprises, in particular inaddition to an Fc moiety as described above, all other parts of theconstant regions, in particular all other parts of the constant regionsof IgG, preferably all other parts of the constant regions of IgG1, morepreferably all other parts of the constant regions of human IgG1.

As outlined above, a particularly preferred antibody according to thepresent invention comprises a (complete) Fc region derived from humanIgG1. More preferably, the antibody according to the present inventioncomprises, in particular in addition to a (complete) Fc region derivedfrom human IgG1 also all other parts of the constant regions of IgG,preferably all other parts of the constant regions of IgG1, morepreferably all other parts of the constant regions of human IgG1.

It is also preferred that the antibody according to the presentinvention, or an antigen binding fragment thereof, binds to 1, 2, 3, 4,5, 6, 7, 8, 9 or 10 of the HBsAg genotypes A, B, C, D, E, F, G, H, I,and J. Examples for the different genotypes of HBsAg include thefollowing: GenBank accession number J02203 (HBV-D, ayws), GenBankaccession number FJ899792.1 (HBV-D, adw2), GenBank accession numberAM282986 (HBV-A), GenBank accession number D23678 (HBV-B1 Japan),GenBank accession number AB117758 (HBV-C1 Cambodia), GenBank accessionnumber AB205192 (HBV-E Ghana), GenBank accession number X69798 (HBV-F4Brazil), GenBank accession number AF160501 (HBV-G USA), GenBankaccession number AY090454 (HBV-H Nicaragua), GenBank accession numberAF241409 (HBV-I Vietnam) and GenBank accession number AB486012 (HBV-JBorneo). The amino acid sequences of the antigenic loop region of the Sdomain of HBsAg of the different genotypes is shown in Table 1 (SEQ IDNO's: 5-15).

More preferably, the antibody according to the present invention, or anantigen binding fragment thereof, binds to at least 6, even morepreferably to at least 8, and particularly preferably to all 10 of theHBsAg genotypes A, B, C, D, E, F, G, H, I, and J. HBV is differentiatedinto many genotypes, according to genome sequence. To date, eightwell-known genotypes (A-H) of the HBV genome have been defined.Moreover, two new genotypes, I and J, have also been identified (SunbulM., 2014, World J Gastroenterol 20(18): 5427-5434). The genotype isknown to affect the progression of the disease and differences betweengenotypes in response to antiviral treatment have been determined. Forexample, genotype A has a tendency for chronicity, whereas viralmutations are frequently encountered in genotype C. Both chronicity andmutation frequency are common in genotype D. Moreover, the genotypes ofHBV are differentially distributed over the world (Sunbul M., 2014,World J Gastroenterol 20(18): 5427-5434). By providing an antibodyaccording to the present invention, or an antigen binding fragmentthereof, which preferably binds to at least 6, preferably to at least 8,more preferably to all 10 of the HBsAg genotypes A, B, C, D, E, F, G, H,I, and J, an antibody is provided, which binds very broadly to thedifferent genotypes.

Preferably, the antibody according to the present invention, or anantigen binding fragment thereof, binds to 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17 or 18 of the HBsAg mutants havingmutations in the antigenic loop region: HBsAg Y100C/P12T, HBsAg P120T,HBsAg P120T/S143L, HBsAg C121S, HBsAg R122D, HBsAg R122I, HBsAg T123N,HBsAg Q129H, HBsAg Q129L, HBsAg M133H, HBsAg M133L, HBsAg M133T, HBsAgK141E, HBsAg P142S, HBsAg S143K, HBsAg D144A, HBsAg G145R and HBsAgN146A. Those mutants are naturally occurring mutants based on the Sdomain of HBsAg Genotype D, Genbank accession no. FJ899792 (SEQ ID NO:4), whereby the mutated amino acid residue(s) are indicated in the name.

SEQ ID NO: 4: MENVTSGFLGPLLVLQAGFFLLTRILTIPQSLDSWWTSLNFLGGTTVCLGQNSQSPTSNHSPTSCPPTCPGYRWMCLRRFIIFLFILLLCLIFLLVLLDYQGMLPVCPLIPGSSTTGTGPCRTCTTPAQGTSMYPSCCCTKPSDGNCTCIPIPSSWAFGKFLWEWASARFSWLSLLVPFVQWFVGLSPTVWLSVIWMMWYWGPSLYSTLSPFLPLLPIFFCLWVYI (the antigenic loop region, i.e. amino acids 101-172, is shown underlined).

The amino acid sequences of the antigenic loop region of the S domain ofHBsAg of the different mutants is shown in Table 1 (SEQ ID NO's: 16-33).

More preferably, the antibody according to the present invention, or anantigen binding fragment thereof, binds to at least 12, even morepreferably to at least 15, and particularly preferably to all 18 of theinfectious HBsAg mutants having mutations in the antigenic loop region:HBsAg Y100C/P120T, HBsAg P120T, HBsAg P120T/S143L, HBsAg C121S, HBsAgR122D, HBsAg R122I, HBsAg T123N, HBsAg Q129H, HBsAg Q129L, HBsAg M133H,HBsAg M133L, HBsAg M133T, HBsAg K141E, HBsAg P142S, HBsAg S143K, HBsAgD144A, HBsAg G145R and HBsAg N146A.

Preferably, the antibody according to the present invention, or anantigen binding fragment thereof, binds to an epitope comprising atleast one, preferably at least two, more preferably at least three aminoacids, even more preferably at least four amino acids of the antigenicloop region of HBsAg, wherein the at least two, preferably the at leastthree, more preferably the at least four amino acids are selected fromamino acids 115-133 of the S domain of HBsAg, preferably from aminoacids 120-133 of the S domain of HBsAg, more preferably from amino acids120-130 of the S domain of HBsAg. Of note, the position of the aminoacids (e.g. 115-133, 120-133, 120-130) refers to the S domain of HBsAgas described above, which is present in all three HBV envelope proteinsS-HBsAg, M-HBsAg, and L-HBsAg, whereby S-HBsAg typically corresponds tothe S domain of HBsAg.

In particular, the antibody according to the present invention, or anantigen binding fragment thereof, binds to an epitope in the antigenicloop region of HBsAg, whereby the epitope is typically formed by one ormore amino acids located at positions selected from amino acid positions115-133, preferably selected from amino acid positions 120-133, morepreferably selected from amino acid positions 120-130 of the S domain ofHBsAg.

The term “formed by” as used herein in the context of an epitope means,that the epitope to which the antibody of the invention, or an antigenbinding fragment thereof, binds to may be linear (continuous) orconformational (discontinuous). A linear or a sequential epitope is anepitope that is recognized by antibodies by its linear sequence of aminoacids, or primary structure. In contrast, a conformational epitope has aspecific three-dimensional shape and protein structure. Accordingly, ifthe epitope is a linear epitope and comprises more than one amino acidlocated at positions selected from amino acid positions 115-133,preferably selected from amino acid positions 120-133 of the S domain ofHBsAg, the amino acids comprised by the epitope are typically located inadjacent positions of the primary structure (i.e. consecutive aminoacids in the amino acid sequence). In the case of a conformationalepitope (3D structure), in contrast, the amino acid sequence typicallyforms a 3D structure as epitope and, thus, the amino acids forming theepitope (or the amino acids “comprised by” the epitope) may be or may benot located in adjacent positions of the primary structure (i.e. maybeor may be not consecutive amino acids in the amino acid sequence).

Preferably, the epitope to which the antibody of the invention, or anantigen binding fragment thereof, binds to is only formed by aminoacid(s) selected from amino acid positions 115-133, preferably selectedfrom amino acid positions 120-133, more preferably selected from aminoacid positions 120-130 of the S domain of HBsAg. In other words, it ispreferred that no (further) amino acids—which are located outside thepositions 115-133, preferably positions 120-133, more preferablypositions 120-130—are required to form the epitope to which the antibodyof the invention, or an antigen binding fragment thereof, binds to.

Preferably, the epitope in the antigenic loop region of HBsAg to whichthe antibody of the invention, or an antigen binding fragment thereof,bind are formed by two or more amino acids located at positions selectedfrom amino acid positions 115-133, preferably selected from amino acidpositions 120-133, more preferably selected from amino acid positions120-130 of the S domain of HBsAg. More preferably, the epitope in theantigenic loop region of HBsAg to which the antibody of the invention,or an antigen binding fragment thereof, bind are formed by three or moreamino acids located at positions selected from amino acid positions115-133, preferably selected from amino acid positions 120-133, morepreferably selected from amino acid positions 120-130 of the S domain ofHBsAg. Even more preferably, the epitope in the antigenic loop region ofHBsAg to which the antibody of the invention, or an antigen bindingfragment thereof, bind are formed by four or more amino acids located atpositions selected from amino acid positions 115-133, preferablyselected from amino acid positions 120-133, more preferably selectedfrom amino acid positions 120-130 of the S domain of HBsAg. In otherwords, it is preferred that, the antibody according to the presentinvention, or an antigen binding fragment thereof, binds to at leastone, preferably at least two, more preferably at least three, even morepreferably to at least four amino acids of the antigenic loop region ofHBsAg selected from amino acid 115-amino acid 133 of the S domain ofHBsAg, preferably from amino acid 120-amino acid 133 of the S domain ofHBsAg, more preferably selected from amino acids 120-130 of the S domainof HBsAg.

More preferably, the antibody according to the present invention, or theantigen binding fragment thereof, binds to an epitope comprising atleast two, preferably at least three, more preferably at least fouramino acids of the antigenic loop region of HBsAg, wherein the at leasttwo, preferably the at least three, more preferably the at least fouramino acids are selected from amino acid 120-amino acid 133, preferablyfrom amino acids 120-130 of The S domain of HBsAg and wherein the atleast two, preferably the at least three, more preferably the at leastfour amino acids are located in adjacent positions (i.e. are consecutiveamino acids in the amino acid sequence/primary structure).

The epitope to which the antibody according to the present invention, oran antigen binding fragment thereof, binds to, is preferably aconformational epitope. Accordingly, it is preferred that the antibodyaccording to the present invention, or an antigen binding fragmentthereof, binds to an epitope comprising at least two, preferably atleast three, more preferably at least four amino acids of the antigenicloop region of HBsAg, wherein the at least two, preferably the at leastthree, more preferably the at least four amino acids are selected fromamino acids 120-133, preferably from amino acids 120-130, of the Sdomain of HBsAg and wherein at least two, preferably at least three, ofthe at least two, preferably the at least three, more preferably the atleast four amino acids are not located in adjacent positions (of theprimary structure).

In other words, (i) either none of the amino acids to which the antibodybinds to (i.e. the amino acids forming the epitope) are located inadjacent positions of the primary structure or (ii) some, for exampletwo or three, of the amino acids to which the antibody binds to (i.e.the amino acids forming the epitope) are located in adjacent positions(of the primary structure) whereas other amino acids to which theantibody binds to (i.e. the amino acids forming the epitope) are notlocated in adjacent positions (of the primary structure).

Amino acids to which the antibody binds to (i.e. the amino acids formingthe epitope), which are not located in adjacent positions of the primarystructure, are typically spaced apart by one or more amino acids, towhich the antibody does not bind to. Preferably, at least one, morepreferably at least two, even more preferably at least three, mostpreferably at least four, particularly preferably at least five aminoacids may be located between the at least two, preferably at leastthree, amino acids, which are comprised by the epitope and which are notlocated in adjacent positions.

Preferably, the antibody according to the present invention, or anantigen binding fragment thereof, binds to an epitope comprising atleast amino acids P120, C121, R122 and C124 of the S domain of HBsAg.More preferably, the antibody or the antigen binding fragment binds toan epitope comprising an amino acid sequence according to SEQ ID NO: 88:

PCRXC

wherein X is any amino acid or no amino acid; preferably X is any aminoacid; more preferably X is T, Y, R, S, or F; even more preferably X isT, Y or R; most preferably X is T or R.

Even more preferably the antibody or the antigen binding fragment bindsto an epitope comprising an amino acid sequence according to SEQ ID NO:80:

TGPCRTC

or to an amino acid sequence sharing at least 80%, preferably at least90%, more preferably at least 95% sequence identity with SEQ ID NO: 80.

Most preferably, the antibody or the antigen binding fragment binds toan epitope comprising an amino acid sequence according to SEQ ID NO: 85:

STTSTGPCRTC

or to an amino acid sequence sharing at least 80%, preferably at least90%, more preferably at least 95% sequence identity with SEQ ID NO: 85.

It is also preferred that the antibody or the antigen binding fragmentbinds to an epitope comprising an amino acid sequence comprising atleast amino acids 145-151 of the S domain of HBsAg:

(SEQ ID NO: 81) GNCTCIP.

More preferably, the antibody or the antigen binding fragment binds toan epitope comprising an amino acid sequence according to SEQ ID NO: 80and an amino acid sequence according to SEQ ID NO: 81.

More preferably, the antibody or the antigen binding fragment binds toan epitope comprising an amino acid sequence according to SEQ ID NO: 85and/or an amino acid sequence according to SEQ ID NO: 87.

As described above, the epitope to which the antibodies of the inventionbinds to may be linear (continuous) or conformational (discontinuous).Preferably, the antibody and antibody fragments of the invention bindsto a conformational epitope, more preferably the conformational epitopeis present only under non-reducing conditions.

However, it is also preferred that the antibody according to the presentinvention, or an antibody fragment thereof, binds to a linear epitope,more preferably the linear epitope is present under both, non-reducingconditions and reducing conditions.

Preferably, the antibody according to the present invention, or anantigen binding fragment thereof, binds to an epitope in the antigenicloop of HBsAg formed by an amino acid sequence according to SEQ ID NO:1:

X₁ X₂ X₃ TC X₄ X₅ X₆A X₇G

wherein X₁, X₂, X₃, X₄, X₅, X₆ and X₇ may be any amino acid (SEQ ID NO:1).

Preferably, X₁, X₂, X₃, X₄, X₅, X₆ and X₇ are amino acids, which areconservatively substituted in comparison to amino acids 120-130 of SEQID NO: 3. It is also preferred that X₁, X₂, X₃, X₄, X₅, X₆ and X, areamino acids, which are conservatively substituted in comparison to aminoacids 20-30 of any of SEQ ID NO's 5-33 (cf. Table 1; referring to theantigenic loop region sequences, i.e. aa 101-172 of the S domain ofdifferent variants of HBsAG).

Preferably, in SEQ ID NO: 1 X₁ is a small amino acid. A “small” aminoacid, as used herein, refers to any amino acid selected from the groupconsisting of alanine, aspartic acid, asparagine, cysteine, glycine,proline, serine, threonine and valine. More preferably, X₁ is proline,serine or threonine.

Preferably, in SEQ ID NO: 1 X₂ is a small amino acid. More preferably,X₂ is cystein or threonine.

Preferably, in SEQ ID NO: 1 X₃ is a charged amino acid or an aliphaticamino acid. A “charged” amino acid, as used herein, refers to any aminoacid selected from the group consisting of arginine, lysine, asparticacid, glutamic acid and histidine. A “aliphatic” amino acid, as usedherein, refers to any amino acid selected from the group consisting ofalanine, glycine, isoleucine, leucine, and valine. More preferably, X₃is arginine, lysine, aspartic acid or isoleucine.

Preferably, in SEQ ID NO: 1 X₄ is a small amino acid and/or ahydrophobic amino acid. A “hydrophobic” amino acid, as used herein,refers to any amino acid selected from the group consisting of alanine,isoleucine, leucine, phenylalanine, valine, tryptophan, tyrosin,methionine, proline and glycine. More preferably, X₄ is methionine orthreonine.

Preferably, in SEQ ID NO: 1 X₅ is a small amino acid and/or ahydrophobic amino acid. More preferably, X₅ is threonine, alanine orisoleucine.

Preferably, in SEQ ID NO: 1 X₆ is a small amino acid and/or ahydrophobic amino acid. More preferably, X₆ is threonine, proline orleucine.

Preferably, in SEQ ID NO: 1 X₇ is a polar amino acid or an aliphaticamino acid. A “polar” amino acid, as used herein, refers to any aminoacid selected from the group consisting of aspartic acid, asparagine,arginine, glutamic acid, histidine, lysine, glutamine, tryptophan,tyrosine, serine, and threonine. More preferably, X₇ is glutamine,histidine or leucine.

Accordingly, it is more preferred that the antibody according to thepresent invention, or an antigen binding fragment thereof, binds to anepitope in the antigenic loop of HBsAg formed by an amino acid sequenceaccording to SEQ ID NO: 2:

X₁ X₂ X₃ TC X₄ X₅ X₆A X₇G

wherein X₁ is P, T or S,

-   -   X₂ is C or S,    -   X₃ is R, K, D or I,    -   X₄ is M or T,    -   X₅ is T, A or I,    -   X₆ is T, P or L, and    -   X₇ is Q, H or L

(SEQ ID NO: 2).

With regard to the preferred epitopes formed by the amino acid sequencesaccording to SEQ ID NO: 1 or 2, it is noted that the term “formed by” asused herein is in particular not intended to imply that the antibodynecessarily binds to each and every amino acid of SEQ ID NO: 1 or 2. Inparticular in the case of the preferred conformational epitope, theantibody may bind only to some of the amino acids of SEQ ID NO: 1 or 2,whereby other amino acid residues may merely act as “spacers”, therebyproviding the 3D structure of the epitope.

Preferably, the antibody according to the present invention, or anantigen binding fragment thereof, binds to an epitope in the antigenicloop of HBsAg formed by one or more, preferably by two or more, morepreferably by three or more and even more preferably by four or moreamino acids of an amino acid sequence selected from SEQ ID NO's 5-33shown below in Table 1.

More preferably, the antibody according to the present invention, or anantigen binding fragment thereof, binds to an antigenic loop region ofHBsAg having an amino acid sequence according to any of SEQ ID NO's 5-33shown below in Table 1 or to a sequence variant thereof. Even morepreferably, the antibody according to the present invention, or anantigen binding fragment thereof, binds to all of the antigenic loopvariants of HBsAg having an amino acid sequence according to any of SEQID NO's 5-33 shown below in Table 1. In other words, it is particularlypreferred if the antibody according to the present invention, or anantigen binding fragment thereof, is able to bind to all of thedifferent antigenic loop regions of HBsAg having an amino acid sequenceaccording to any of SEQ ID NO's 5-33.

TABLE 1 Exemplary amino acid sequences of the antigenic loop regionof the S domain of HBsAg (residues 101-172 of the S domainof HBsAg-except for SEQ ID NO: 16 which refers to residues100-172 of the S domain of HBsAg in order to include therelevant mutation) of the different genotypes and mutantsas used herein. SEQ ID Name NO. Amino acid sequence J02203 (D, ayw3) 5QGMLPVCPLIPGSSTTSTGPCRTCMTTAQGTSMYPSCCCTKPSDGNCTCIPIPSSWAFGKFLWEWASARFSW FJ899792 (D, 6QGMLPVCPLIPGSSTTGTGPCRTCTTPAQGTSM adw2)YPSCCCTKPSDGNCTCIPIPSSWAFGKFLWEWAS ARFSW AM282986 7QGMLPVCPLIPGTTTTSTGPCKTCTTPAQGNSMFPSCCC (A)TKPSDGNCTCIPIPSSWAFAKYLWEWASVRFSW D23678 (B1) 8QGMLPVCPLIPGSSTTSTGPCKTCTTPAQGTSMFPSCCCTKPTDGNCTCIPIPSSWAFAKYLWEWASVRFSW AB117758 (C1) 9QGMLPVCPLLPGTSTTSTGPCKTCTIPAQGTSMFPSCCCTKPSDGNCTCIPIPSSWAFARFLWEWASVRFSW AB205192 (E) 10QGMLPVCPLIPGSSTTSTGPCRTCTTLAQGTSMFPSCCCSKPSDGNCTCIPIPSSWAFGKFLWEWASARFSW X69798 (F4) 11QGMLPVCPLLPGSTTTSTGPCKTCTTLAQGTSMFPSCCCSKPSDGNCTCIPIPSSWALGKYLWEWASARFSW AF160501 (G) 12QGMLPVCPLIPGSSTTSTGPCKTCTTPAQGNSMYPSCCCTKPSDGNCTCIPIPSSWAFAKYLWEWASVRFSW AY090454 (H) 13QGMLPVCPLLPGSTTTSTGPCKTCTTLAQGTSMFPSCCCTKPSDGNCTCIPIPSSWAFGKYLWEWASARFSW AF241409 (I) 14QGMLPVCPLIPGSSTTSTGPCKTCTTPAQGNSMYPSCCCTKPSDGNCTCIPIPSSWAFAKYLWEWASARFSW AB486012 (J) 15QGMLPVCPLLPGSTTTSTGPCRTCTITAQGTSMFPSCCCTKPSDGNCTCIPIPSSWAFAKFLWEWASVRFSW HBsAg 16CQGMLPVCPLIPGSSTTGTGTCRTCTTPAQGTSMYPSCC Y100C/P120TCTKPSDGNCTCIPIPSSWAFGKFLWEWASARFSW HBsAg P120T 17QGMLPVCPLIPGSSTTGTGTCRTCTTPAQGTSMYPSCCCTKPSDGNCTCIPIPSSWAFGKFLWEWASARFSW HBsAg 18QGMLPVCPLIPGSSTTGTGTCRTCTTPAQGTSMYPSCCC P120T/S143LTKPLDGNCTCIPIPSSWAFGKFLWEWASARFSW HBsAg C121S 19QGMLPVCPLIPGSSTTGTGPSRTCTTPAQGTSMYPSCCCTKPSDGNCTCIPIPSSWAFGKFLWEWASARFSW HBsAg R122D 20QGMLPVCPLIPGSSTTGTGPCDTCTTPAQGTSMYPSCCCTKPSDGNCTCIPIPSSWAFGKFLWEWASARFSW HBsAg R122I 21QGMLPVCPLIPGSSTTGTGPCITCTTPAQGTSMYPSCCCTKPSDGNCTCIPIPSSWAFGKFLWEWASARFSW HBsAg T123N 22QGMLPVCPLIPGSSTTGTGPCRNCTTPAQGTSMYPSCCCTKPSDGNCTCIPIPSSWAFGKFLWEWASARFSW HBsAg Q129H 23QGMLPVCPLIPGSSTTGTGPCRTCTTPAHGTSMYPSCCCTKPSDGNCTCIPIPSSWAFGKFLWEWASARFSW HBsAg Q129L 24QGMLPVCPLIPGSSTTGTGPCRTCTTPALGTSMYPSCCCTKPSDGNCTCIPIPSSWAFGKFLWEWASARFSW HBsAg M133H 25QGMLPVCPLIPGSSTTGTGPCRTCTTPAQGTSHYPSCCCTKPSDGNCTCIPIPSSWAFGKFLWEWASARFSW HBsAg M133L 26QGMLPVCPLIPGSSTTGTGPCRTCTTPAQGTSLYPSCCCTKPSDGNCTCIPIPSSWAFGKFLWEWASARFSW HBsAg M133T 27QGMLPVCPLIPGSSTTGTGPCRTCTTPAQGTSTYPSCCCTKPSDGNCTCIPIPSSWAFGKFLWEWASARFSW HBsAg K141E 28QGMLPVCPLIPGSSTTGTGPCRTCTTPAQGTSMYPSCCCTEPSDGNCTCIPIPSSWAFGKFLWEWASARFSW HBsAg P142S 29QGMLPVCPLIPGSSTTGTGPCRTCTTPAQGTSMYPSCCCTKSSDGNCTCIPIPSSWAFGKFLWEWASARFSW HBsAg S143K 30QGMLPVCPLIPGSSTTGTGPCRTCTTPAQGTSMYPSCCCTKPKDGNCTCIPIPSSWAFGKFLWEWASARFSW HBsAg D144A 31QGMLPVCPLIPGSSTTGTGPCRTCTTPAQGTSMYPSCCCTKPSAGNCTCIPIPSSWAFGKFLWEWASARFSW HBsAg G145R 32QGMLPVCPLIPGSSTTGTGPCRTCTTPAQGTSMYPSCCCTKPSDRNCTCIPIPSSWAFGKFLWEWASARFSW HBsAg N146A 33QGMLPVCPLIPGSSTTGTGPCRTCTTPAQGTSMYPSCCCTKPSDGACTCIPIPSSWAFGKFLWEWASARFSW

In general, the antibody according to the present invention, or theantigen binding fragment thereof, preferably comprises (at least) threecomplementarity determining regions (CDRs) on a heavy chain and (atleast) three CDRs on alight chain. In general, complementaritydetermining regions (CDRs) are the hypervariable regions present inheavy chain variable domains and light chain variable domains.Typically, the CDRs of a heavy chain and the connected light chain of anantibody together form the antigen receptor. Usually, the three CDRs(CDR1, CDR2, and CDR3) are arranged non-consecutively in the variabledomain. Since antigen receptors are typically composed of two variabledomains (on two different polypeptide chains, i.e. heavy and lightchain), there are six CDRs for each antigen receptor (heavy chain:CDRH1, CDRH2, and CDRH3; light chain: CDRL1, CDRL2, and CDRL3). A singleantibody molecule usually has two antigen receptors and thereforecontains twelve CDRs. The CDRs on the heavy and/or light chain may beseparated by framework regions, whereby a framework region (FR) is aregion in the variable domain which is less “variable” than the CDR. Forexample, a chain (or each chain, respectively) may be composed of fourframework regions, separated by three CDR's.

The sequences of the heavy chains and light chains of an exemplaryantibody of the invention, comprising three different CDRs on the heavychain and three different CDRs on the light chain were determined. Theposition of the CDR amino acids are defined according to the IMGTnumbering system (IMGT: http://www.imgt.org/; cf. Lefranc, M.-P. et al.(2009) Nucleic Acids Res. 37, D1006-D1012).

Table 2 shows the amino acid sequences of the CDR's and the heavy chainvariable region (VH) and the light chain variable region (VL) of anexemplary antibody according to the present invention (“HBC34”):

SEQ ID HBC34 NO. Amino acid sequence CDRH1 34 GRIFRSFY CDRH2 35 NQDGSEKCDRH2 66 INQDGSEK long CDRH3 36 AAWSGNSGGMDV CDRL1 37 KLGNKN CDRL2 38EVK CDRL2 39 VIYEVKYRP long CDRL3 40 QTWDSTTVV VH 41ELQLVESGGGWVQPGGSQRLSCAASGRIFRSFYMSWVRQAPGKGLEWVATINQDGSEKLYVDSVKGRFTISRDNAKNSLFLQMNNLRVEDTAVYYCAAWSGNSGGMDVWGQGTTVSVSS VL 42SYELTQPPSVSVSPGQTVSIPCSGDKLGNKNVCWFQHKPGQSPVLVIYEVKYRPSGIPERFSGSNSGNTATLTISGTQAMDE AAYFCQTWDSTTVVFGGGTRLTVL

It is thus preferred that the antibody, or the antigen binding fragmentthereof, according to the present invention comprises amino acidsequences having at least 70%, at least 75%, at least 80%, at least 85%,at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% identity to at least one of theCDR sequences, the VH sequence and/or the VL sequence shown in Table 2.

Table 3 shows the amino acid sequences of the CDR's and the heavy chainvariable region (VH) and the light chain variable region (VL) of afurther exemplary antibody according to the present invention(“HBC34v7”):

SEQ ID HBC34v7 NO. Amino acid sequence CDRH1 34 GRIFRSFY CDRH2 35NQDGSEK CDRH2 66 INQDGSEK long CDRH3 36 AAWSGNSGGMDV CDRL1 37 KLGNKNCDRL2 38 EVK CDRL2 39 VIYEVKYRP long CDRL3 58 QTFDSTTVV VH 41ELQLVESGGGWVQPGGSQRLSCAASGRIFRSFYMSWVRQAPGKGLEWVATINQDGSEKLYVDSVKGRFTISRDNAKNSLFLQMNNLRVEDTAVYYCAAWSGNSGGMDVWGQGTTVSV SS VL 59SYELTQPPSVSVSPGQTVSIPCSGDKLGNKNVCWFQHKPGQSPVLVIYEVKYRPSGIPERFSGSNSGNTATLTISGTQ AMDEAAYFCQTFDSTTVVFGGGTRLTVL

It is thus also preferred that the antibody, or the antigen bindingfragment thereof, according to the present invention comprises aminoacid sequences having at least 70%, at least 75%, at least 80%, at least85%, at least 88%, at least 90%, at least 92%, at least 95%, at least96%, at least 97%, at least 98% or at least 99% identity to at least oneof the CDR sequences, the VH sequence and/or the VL sequence shown inTable 3.

Table 4 shows the amino acid sequences of the CDR's and the heavy chainvariable region (VH) and the light chain variable region (VL) of afurther exemplary antibody according to the present invention(“HBC34v23”):

SEQ ID HBC34v23 NO. Amino acid sequence CDRH1 34 GRIFRSFY CDRH2 35NQDGSEK CDRH2 66 INQDGSEK long CDRH3 36 AAWSGNSGGMDV CDRL1 37 KLGNKNCDRL2 38 EVK CDRL2 39 VIYEVKYRP long CDRL3 58 QTFDSTTVV VH 41ELQLVESGGGWVQPGGSQRLSCAASGRIFRSFYMSWVRQAPGKGLEWVATINQDGSEKLYVDSVKGRFTISRDNAKNSLFLQMNNLRVEDTAVYYCAAWSGNSGGMDVWGQGTT VSVSS VL 65SYELTQPPSVSVSPGQTASITCSGDKLGNKNACWYQQKPGQSPVLVIYEVKYRPSGIPERFSGSNSGNTATLTISG TQAMDEADYYCQTFDSTTVVFGGGTKLTVL

It is thus also preferred that the antibody, or the antigen bindingfragment thereof, according to the present invention comprises aminoacid sequences having at least 70%, at least 75%, at least 80%, at least85%, at least 88%, at least 90%, at least 92%, at least 95%, at least96%, at least 97%, at least 98% or at least 99% identity to at least oneof the CDR sequences, the VH sequence and/or the VL sequence shown inTable 4.

Table 5 shows the amino acid sequences of the CDR's and the heavy chainvariable region (VH) and the light chain variable region (VL) of afurther exemplary antibody according to the present invention(“HBC34v31”):

SEQ ID HBC34v31 NO. Amino acid sequence CDRH1 34 GRIFRSFY CDRH2 35NQDGSEK CDRH2 66 INQDGSEK long CDRH3 36 AAWSGNSGGMDV CDRL1 37 KLGNKNCDRL2 38 EVK CDRL2 39 VIYEVKYRP long CDRL3 40 QTWDSTTVV VH 67EVQLVESGGGLVQPGGSLRLSCAASGRIFRSFYMSWVRQAPGKGLEWVANINQDGSEKLYVDSVKGRFTISRDNAKNSLFLQMNNLRVEDTAVYYCAAWSGNSGGMDVWGQGTT VTVSS VL 42SYELTQPPSVSVSPGQTVSIPCSGDKLGNKNVCWFQHKPGQSPVLVIYEVKYRPSGIPERFSGSNSGNTATLTISG TQAMDEAAYFCQTWDSTTVVFGGGTRLTVL

It is thus preferred that the antibody, or the antigen binding fragmentthereof, according to the present invention comprises amino acidsequences having at least 70%, at least 75%, at least 80%, at least 85%,at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% identity to at least one of theCDR sequences, the VH sequence and/or the VL sequence shown in Table 5.

Table 6 shows the amino acid sequences of the CDR's and the heavy chainvariable region (VH) and the light chain variable region (VL) of afurther exemplary antibody according to the present invention(“HBC34v32”):

SEQ ID HBC34v32 NO. Amino acid sequence CDRH1 34 GRIFRSFY CDRH2 35NQDGSEK CDRH2 66 INQDGSEK long CDRH3 36 AAWSGNSGGMDV CDRL1 37 KLGNKNCDRL2 38 EVK CDRL2 39 VIYEVKYRP long CDRL3 58 QTFDSTTVV VH 67EVQLVESGGGLVQPGGSLRLSCAASGRIFRSFYMSWVRQAPGKGLEWVANINQDGSEKLYVDSVKGRFTISRDNAKNSLFLQMNNLRVEDTAVYYCAAWSGNSGGMDVWGQGTT VTVSS VL 59SYELTQPPSVSVSPGQTVSIPCSGDKLGNKNVCWFQHKPGQSPVLVIYEVKYRPSGIPERFSGSNSGNTATLTISG TQAMDEAAYFCQTFDSTTVVFGGGTRLTVL

It is thus also preferred that the antibody, or the antigen bindingfragment thereof, according to the present invention comprises aminoacid sequences having at least 70%, at least 75%, at least 80%, at least85%, at least 88%, at least 90%, at least 92%, at least 95%, at least96%, at least 97%, at least 98% or at least 99% identity to at least oneof the CDR sequences, the VH sequence and/or the VL sequence shown inTable 6.

Table 7 shows the amino acid sequences of the CDR's and the heavy chainvariable region (VH) and the light chain variable region (VL) of afurther exemplary antibody according to the present invention(“HBC34v33”):

SEQ ID HBC34v33 NO. Amino acid sequence CDRH1 34 GRIFRSFY CDRH2 35NQDGSEK CDRH2 66 INQDGSEK long CDRH3 36 AAWSGNSGGMDV CDRL1 37 KLGNKNCDRL2 38 EVK CDRL2 39 VIYEVKYRP long CDRL3 58 QTFDSTTVV VH 67EVQLVESGGGLVQPGGSLRLSCAASGRIFRSFYMSWVRQAPGKGLEWVANINQDGSEKLYVDSVKGRFTISRDNAKNSLFLQMNNLRVEDTAVYYCAAWSGNSGGMDVWGQGTT VTVSS VL 65SYELTQPPSVSVSPGQTASITCSGDKLGNKNACWYQQKPGQSPVLVIYEVKYRPSGIPERFSGSNSGNTATLTISG TQAMDEADYYCQTFDSTTVVFGGGTKLTVL

It is thus also preferred that the antibody, or the antigen bindingfragment thereof, according the present invention comprises amino acidsequences having at least 70%, at least 75%, at least 80%, at least 85%,at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, atleast 97%, at least 98% or at least 99% identity to at least one of theCDR sequences, the VH sequence and/or the VL sequence shown in Table 7.

Table 8 provides an overview over the VH and VL modifications of theexemplary antibody variants “HBC34v7”, “HBC34v23”, “HBC34v31”,“HBC34v32” and “HBC34v33” of the wild-type (WT) antibody “HBC34” andover the respective SEQ ID NOs of the corresponding CDR and VH/VL aminoacid sequences:

Variant VH VL CDR name modification modification H1 H2 H3 L1 L2 L3 VL VHHBC34 WT WT 34 35/66 36 37 38/39 40 41 42 HBC34-V7 WT W107F 34 35/66 3637 38/39 58 41 59 HBC34-V23 WT W07F/ 34 35/66 36 37 38/39 58 41 65FR1234GL/ CDR2Y66 HBC34-V31 FR124GL WT 34 35/66 36 37 38/39 40 67 42HBC34-V32 FR124GL W107F 34 35/66 36 37 38/39 58 67 59 HBC34-V33 FR124GLW07F/ 34 35/66 36 37 38/39 58 67 65 FR1234GL/ CDR2Y66

Preferably, the antibody, or the antigen binding fragment thereofaccording to the present invention comprises a heavy chain comprising atleast one CDRH1, at least one CDRH2 and at least one CDRH3 and alightchain comprising at least one CDRL1, at least one CDRL2 and at least oneCDRL3, wherein at least one CDR, preferably the at least one heavy chainCDRH3, comprises or consists of an amino acid sequence according to SEQID NO: 36 or a functional sequence variant thereof as described herein.Accordingly, it is preferred that the at least one heavy chain CDRH3comprises an amino acid sequence sharing at least 80%, preferably atleast 85%, more preferably at least 90%, even more preferably at least95% and particularly preferably at least 98% or 99% sequence identitywith SEQ ID NO: 36. More preferably, the at least one heavy chain CDRH3comprises an amino acid sequence according to SEQ ID NO: 36.

It is also preferred that the antibody or antigen binding fragmentcomprises a heavy chain comprising at least one CDRH1, at least oneCDRH2 and at least one CDRH3 and a light chain comprising at least oneCDRL1, at least one CDRL2 and at least one CDRL3, wherein the at leastone light chain CDRL3 comprises an amino acid sequence according to SEQID NO: 40 or SEQ ID NO: 58, or a functional sequence variant thereof.Accordingly, it is preferred that the at least one light chain CDRL3comprises an amino acid sequence sharing at least 80%, preferably atleast 85%, more preferably at least 90%, even more preferably at least95% and particularly preferably at least 98% or 99% sequence identitywith SEQ ID NO: 40 or SEQ ID NO: 58. More preferably, the at least onelight chain CDRL3 comprises an amino acid sequence according to SEQ IDNO: 40 or SEQ ID NO: 58.

More preferably, the antibody, or the antigen binding fragment thereof,according to the present invention comprises a heavy chain comprising atleast one CDRH1, at least one CDRH2 and at least one CDRH3 and a lightchain comprising at least one CDRL1, at least one CDRL2 and at least oneCDRL3, wherein the at least one heavy chain CDRH1 comprises an aminoacid sequence according to SEQ ID NO: 34 or a functional sequencevariant thereof, the at least one CDRH2 comprises an amino acid sequenceaccording to SEQ ID NO: 35 or a functional sequence variant thereofand/or the at least one heavy chain CDRH3 comprises an amino acidsequence according to SEQ ID NO: 36 or a functional sequence variantthereof. More preferably, the antibody, or the antigen binding fragmentthereof, according to the present invention comprises a heavy chaincomprising at least one CDRH1, at least one CDRH2 and at least one CDRH3and a light chain comprising at least one CDRL1, at least one CDRL2 andat least one CDRL3, wherein the at least one heavy chain CDRH1 comprisesan amino acid sequence according to SEQ ID NO: 34 or a functionalsequence variant thereof, the at least one CDRH2 comprises an amino acidsequence according to SEQ ID NO: 35 or 66 or a functional sequencevariant thereof and/or the at least one heavy chain CDRH3 comprises anamino acid sequence according to SEQ ID NO: 36 or a functional sequencevariant thereof. Accordingly, it is preferred that the at least oneheavy chain CDRH1 comprises an amino acid sequence sharing at least 80%,preferably at least 85%, more preferably at least 90%, even morepreferably at least 95% and particularly preferably at least 98% or 99%sequence identity with SEQ ID NO: 34; the at least one heavy chain CDRH2comprises an amino acid sequence sharing at least 80%, preferably atleast 85%, more preferably at least 90%, even more preferably at least95% and particularly preferably at least 98% or 99% sequence identitywith SEQ ID NO: 35 or 66; and/or the at least one heavy chain CDRH3comprises an amino acid sequence sharing at least 80%, preferably atleast 85%, more preferably at least 90%, even more preferably at least95% and particularly preferably at least 98% or 99% sequence identitywith SEQ ID NO: 36. More preferably, the at least one heavy chain CDRH1comprises an amino acid sequence according to SEQ ID NO: 34; the atleast one heavy chain CDRH2 comprises an amino acid sequence accordingto SEQ ID NO: 35 or 66; and/or the at least one heavy chain CDRH3comprises an amino acid sequence according to SEQ ID NO: 36.

Preferably, the antibody, or the antigen binding fragment thereof,according to the present invention comprises a heavy chain comprising atleast one CDRH1, at least one CDRH2 and at least one CDRH3 and a lightchain comprising at least one CDRL1, at least one CDRL2 and at least oneCDRL3, wherein the at least one CDRL1 comprises an amino acid sequenceaccording to SEQ ID NO: 37 or a functional sequence variant thereof, theat least one CDRL2 comprises an amino acid sequence according to SEQ IDNO: 38 or 39 or a functional sequence variant thereof, and/or the atleast one CDRL3 amino comprises an amino acid sequence according to SEQID NO: 40 or a functional sequence variant thereof. It is also preferredthat the antibody, or the antigen binding fragment thereof, according tothe present invention comprises a heavy chain comprising at least oneCDRH1, at least one CDRH2 and at least one CDRH3 and a light chaincomprising at least one CDRL1, at least one CDRL2 and at least oneCDRL3, wherein the at least one light chain CDRL1 comprises an aminoacid sequence according to SEQ ID NO: 37 or a functional sequencevariant thereof, the at least one light chain CDRL2 comprises an aminoacid sequence according to SEQ ID NO: 38 or 39 or a functional sequencevariant thereof, and/or the at least one light chain CDRL3 aminocomprising an amino acid sequence according to SEQ ID NO: 58 or afunctional sequence variant thereof. Accordingly, it is preferred thatthe at least one light chain CDRL1 comprises an amino acid sequencesharing at least 80%, preferably at least 85%, more preferably at least90%, even more preferably at least 95% and particularly preferably atleast 98% or 99% sequence identity with SEQ ID NO: 37; the at least onelight chain CDRL2 comprises an amino acid sequence sharing at least 80%,preferably at least 85%, more preferably at least 90%, even morepreferably at least 95% and particularly preferably at least 98% or 99%sequence identity with SEQ ID NO: 38 or 39; and/or the at least onelight chain CDRL3 comprises an amino acid sequence sharing at least 80%,preferably at least 85%, more preferably at least 90%, even morepreferably at least 95% and particularly preferably at least 98% or 99%sequence identity with SEQ ID NO: 40 or 58. More preferably, the atleast one light chain CDRL1 comprises an amino acid sequence accordingto SEQ ID NO: 37; the at least one light chain CDRL2 comprises an aminoacid sequence according to SEQ ID NO: 38 or 39; and/or the at least onelight chain CDRL3 comprises an amino acid sequence according to SEQ IDNO: 40 or 58.

Accordingly, it is also preferred that the antibody, or the antigenbinding fragment thereof, according to the present invention comprisesCDRH1, CDRH2, and CDRH3 amino acid sequences and CDRL1, CDRL2, and CDRL3amino acid sequences according to SEQ ID NOs: 34-38 and 40 or functionalsequence variants thereof or according to SEQ ID NOs: 34-37 and 39-40 orfunctional sequence variants thereof, respectively. It is also preferredthat the antibody or the antigen binding fragment comprises CDRH1,CDRH2, and CDRH3 amino acid sequences and CDRL1, CDRL2, and CDRL3 aminoacid sequences according to SEQ ID NOs: 34, 36-38, 40 and 66 orfunctional sequence variants thereof or according to SEQ ID NOs: 34,36-37, 39-40 and 66 or functional sequence variants thereof,respectively. It is also preferred that the antibody or the antigenbinding fragment comprises CDRH1, CDRH2, and CDRH3 amino acid sequencesand CDRL1, CDRL2, and CDRL3 amino acid sequences according to SEQ IDNOs: 34-38 and 58 or functional sequence variants thereof or accordingto SEQ ID NOs: 34-37, 39 and 58 or functional sequence variants thereof,respectively. It is furthermore preferred that the antibody or theantigen binding fragment comprises CDRH1, CDRH2, and CDRH3 amino acidsequences and CDRL1, CDRL2, and CDRL3 amino acid sequences according toSEQ ID NOs: 34, 36-38, 58 and 66 or functional sequence variants thereofor according to SEQ ID NOs: 34, 36-37, 39, 58 and 66 or functionalsequence variants thereof, respectively.

Accordingly, it is preferred that the antibody or the antigen bindingfragment comprises CDRH1, CDRH2, and CDRH3 amino acid sequences andCDRL1, CDRL2, and CDRL3 amino acid sequences (i) sharing at least 80%,preferably at least 85%, more preferably at least 90%, even morepreferably at least 95% and particularly preferably at least 98% or 99%sequence identity with each of SEQ ID NOs: 34-38 and 40, respectively;or (ii) sharing at least 80%, preferably at least 85%, more preferablyat least 90%, even more preferably at least 95% and particularlypreferably at least 98% or 99% sequence identity with each of SEQ IDNOs: 34-37, 39 and 40, respectively; or (iii) sharing at least 80%,preferably at least 85%, more preferably at least 90%, even morepreferably at least 95% and particularly preferably at least 98% or 99%sequence identity with each of SEQ ID NOs: 34-38 and 58, respectively;or (iv) sharing at least 80%, preferably at least 85%, more preferablyat least 90%, even more preferably at least 95% and particularlypreferably at least 98% or 99% sequence identity with each of SEQ IDNOs: 34-37, 39 and 58, respectively; or (v) sharing at least 80%,preferably at least 85%, more preferably at least 90%, even morepreferably at least 95% and particularly preferably at least 98% or 99%sequence identity with each of SEQ ID NOs: 34, 36-38, 40 and 66,respectively; or (vi) sharing at least 80%, preferably at least 85%,more preferably at least 90%, even more preferably at least 95% andparticularly preferably at least 98% or 99% sequence identity with eachof SEQ ID NOs: 34, 36-37, 39, 40 and 66, respectively; or (vii) sharingat least 80%, preferably at least 85%, more preferably at least 90%,even more preferably at least 95% and particularly preferably at least98% or 99% sequence identity with each of SEQ ID NOs: 34, 36-38, 58 and66, respectively; or (viii) sharing at least 80%, preferably at least85%, more preferably at least 90%, even more preferably at least 95% andparticularly preferably at least 98% or 99% sequence identity with eachof SEQ ID NOs: 34, 36-37, 39, 58 and 66, respectively.

More preferably, the antibody or the antigen binding fragment comprisesCDRH1, CDRH2, and CDRH3 amino acid sequences and CDRL1, CDRL2, and CDRL3amino acid sequences (i) according to SEQ ID NOs: 34-38 and 40,respectively; or (ii) according to SEQ ID NOs: 34-37, 39 and 40,respectively; or (iii) according to SEQ ID NOs: 34-38 and 58,respectively; or (iv) according to SEQ ID NOs: 34-37, 39 and 58,respectively; or (v) according to SEQ ID NOs: 34, 36-38, 40 and 66,respectively; or (vi) according to SEQ ID NOs: 34, 36-37, 39, 40 and 66,respectively; or (vii) according to SEQ ID NOs: 34, 36-38, 58 and 66,respectively; or (viii) according to SEQ ID NOs: 34, 36-37, 39, 58 and66, respectively.

Moreover, it is also preferred that the antibody, or the antigen bindingfragment thereof, according to the present invention comprises a heavychain variable region (VH) amino acid sequence according to SEQ ID NO:41 or a functional sequence variant thereof and/or a light chainvariable region (VL) amino acid sequence according to SEQ ID NO: 42 or afunctional sequence variant thereof. Accordingly, it is preferred thatthe antibody or the antigen binding fragment comprises a heavy chainvariable region (VH) amino acid sequence sharing at least 80%,preferably at least 85%, more preferably at least 90%, even morepreferably at least 95% and particularly preferably at least 98% or 99%sequence identity with SEQ ID NO: 41 and a light chain variable region(VL) amino acid sequence sharing at least 80%, preferably at least 85%,more preferably at least 90%, even more preferably at least 95% andparticularly preferably at least 98% or 99% sequence identity with SEQID NO: 42. More preferably, the antibody or the antigen binding fragmentcomprises a heavy chain variable region (VH) amino acid sequenceaccording to SEQ ID NO: 41 and a light chain variable region (VL) aminoacid sequence according to SEQ ID NO: 42.

Moreover, it is also preferred that the antibody, or the antigen bindingfragment thereof, according to the present invention comprises a heavychain variable region (VH) amino acid sequence according to SEQ ID NO:41 or 67 or a functional sequence variant thereof and/or a light chainvariable region (VL) amino acid sequence according to SEQ ID NO: 42 or afunctional sequence variant thereof. Accordingly, it is preferred thatthe antibody or the antigen binding fragment comprises a heavy chainvariable region (VH) amino acid sequence sharing at least 80%,preferably at least 85%, more preferably at least 90%, even morepreferably at least 95% and particularly preferably at least 98% or 99%sequence identity with SEQ ID NO: 41 or 67 and a light chain variableregion (VL) amino acid sequence sharing at least 80%, preferably atleast 85%, more preferably at least 90%, even more preferably at least95% and particularly preferably at least 98% or 99% sequence identitywith SEQ ID NO: 42. More preferably, the antibody or the antigen bindingfragment comprises a heavy chain variable region (VH) amino acidsequence according to SEQ ID NO: 41 or 67 and a light chain variableregion (VL) amino acid sequence according to SEQ ID NO: 42.

Preferably, the antibody or the antigen binding fragment comprises aheavy chain variable region (VH) amino acid sequence according to SEQ IDNO: 41 or 67 or a functional sequence variant thereof and a light chainvariable region (VL) amino acid sequence according to SEQ ID NO: 59 or65 or a functional sequence variant thereof. Accordingly, it ispreferred that the antibody or the antigen binding fragment comprises(i) a heavy chain variable region (VH) amino acid sequence sharing atleast 80%, preferably at least 85%, more preferably at least 90%, evenmore preferably at least 95% and particularly preferably at least 98% or99% sequence identity with SEQ ID NO: 41 or 67; and (ii) a light chainvariable region (VL) amino acid sequence sharing at least 80%,preferably at least 85%, more preferably at least 90%, even morepreferably at least 95% and particularly preferably at least 98% or 99%sequence identity with SEQ ID NO: 59 or 65. More preferably, theantibody or the antigen binding fragment comprises a heavy chainvariable region (VH) amino acid sequence according to SEQ ID NO: 41 or67 and a light chain variable region (VL) amino acid sequence accordingto SEQ ID NO: 59 or 65.

Preferably, the antibody, or the antigen binding fragment thereof,according to the present invention comprises at least one heavy chainCDRH1, CDRH2 and CDRH3 and light chain CDRL1, CDRL2, and CDRL3 aminoacid sequences that are at least 80%, for example, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or 100% identical to the amino acid sequences of SEQ ID NO's:34-38 and 40 or to the amino acid sequences of SEQ ID NOs: 34-37 and39-40, respectively.

Preferably, the antibody, or the antigen binding fragment thereof,according to the present invention comprises at least one heavy chainCDRH1, CDRH2 and CDRH3 and light chain CDRL1, CDRL2, and CDRL3 aminoacid sequences that are at least 80%, for example, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or 100% identical to the amino acid sequences of SEQ ID NO's:34-38 and 58, respectively, or to the amino acid sequences of SEQ IDNOs: 34-37, 39 and 58, respectively. Preferably, the antibody, or theantigen binding fragment thereof, according to the present inventioncomprises at least one heavy chain CDRH1, CDRH2 and CDRH3 and lightchain CDRL1, CDRL2, and CDRL3 amino acid sequences that are at least80%, for example, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to theamino acid sequences of SEQ ID NO's: 34, 36-38, 40 and 66, respectively,or to the amino acid sequences of SEQ ID NOs: 34, 36-37, 39-40 and 66,respectively. Preferably, the antibody, or the antigen binding fragmentthereof, according to the present invention comprises at least one heavychain CDRH1, CDRH2 and CDRH3 and light chain CDRL1, CDRL2, and CDRL3amino acid sequences that are at least 80%, for example, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or 100% identical to the amino acid sequences of SEQ IDNO's: 34, 36-38, 58 and 66, respectively, or to the amino acid sequencesof SEQ ID NOs: 34, 36-37, 39, 58 and 66, respectively.

It is also preferred that the antibody, or the antigen binding fragmentthereof, according to the present invention comprises at least one heavychain variable region (VH) and at least one light chain variable region(VL) amino acid sequence that is at least 80%, for example, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100% identical to the amino acid sequences of SEQID NOs: 41-42.

It is also preferred that the antibody, or the antigen binding fragmentthereof, according to the present invention comprises at least one heavychain variable region (VH) and at least one light chain variable region(VL) amino acid sequence that is at least 80%, for example, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100% identical to the amino acid sequences of SEQID NOs: 41 and 59.

It is also preferred that the antibody, or the antigen binding fragmentthereof, according to the present invention comprises at least one heavychain variable region (VH) and at least one light chain variable region(VL) amino acid sequence that is at least 80%, for example, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100% identical to the amino acid sequences of SEQID NOs: 41 and 65.

Particularly preferably, the antibody, or the antigen binding fragmentthereof, according to the present invention is gHBC34, in particular,the antibody, or the antigen binding fragment thereof, according to thepresent invention is HBC34.

The present inventors have isolated a monoclonal antibody (mAb)according to the present invention, which is referred to herein as HBC34(cf. Example 1). Based on that antibody HBC34, in particular on the VHand VL genes of HBC34, the term “gHBC34”, as used herein, refer to arespective “generic” antibody, or antigen binding fragments thereof.Namely, “gHBC34” refers to an antibody, or antigen binding fragmentthereof, having a CDRH1 amino acid sequence according to SEQ ID NO: 34,a CDRH2 amino acid sequence according to SEQ ID NO: 35, a CDRH3 aminoacid sequence according to SEQ ID NO: 36, a CDRL1 amino acid sequenceaccording to SEQ ID NO: 37, a CDRL2 amino acid sequence according to SEQID NO: 38 or 39, and a CDRL3 amino acid sequence according to SEQ ID NO:40. In particular, “gHBC34” refers to an antibody, or antigen bindingfragment thereof, having a CDRH1 amino acid sequence according to SEQ IDNO: 34, a CDRH2 amino acid sequence according to SEQ ID NO: 35 or 66, aCDRH3 amino acid sequence according to SEQ ID NO: 36, a CDRL1 amino acidsequence according to SEQ ID NO: 37, a CDRL2 amino acid sequenceaccording to SEQ ID NO: 38 or 39, and a CDRL3 amino acid sequenceaccording to SEQ ID NO: 40. The heavy chain variable region (V_(H)) of“gHBC34” has an amino acid sequence according to SEQ ID NO: 41 and thelight chain variable region (V_(L)) of “gHBC34” has an amino acidsequence according to SEQ ID NO: 42.

Particularly preferably, the antibody, or the antigen binding fragmentthereof, according to the present invention is gHBC34v7, in particular,the antibody, or the antigen binding fragment thereof, according to thepresent invention is HBC34v7.

The present inventors have identified a further monoclonal antibody(mAb) according to the present invention, which is referred to herein asHBC34v7 (cf. Example 11). Based on that antibody HBC34v7, in particularon the VH and VL genes of HBC34v7, the term “gHBC34v7”, as used herein,refer to a respective “generic” antibody, or antigen binding fragmentsthereof. Namely, “gHBC34v7” refers to an antibody, or antigen bindingfragment thereof, having a CDRH1 amino acid sequence according to SEQ IDNO: 34, a CDRH2 amino acid sequence according to SEQ ID NO: 35 or 66, aCDRH3 amino acid sequence according to SEQ ID NO: 36, a CDRL1 amino acidsequence according to SEQ ID NO: 37, a CDRL2 amino acid sequenceaccording to SEQ ID NO: 38 or 39, and a CDRL3 amino acid sequenceaccording to SEQ ID NO: 58. The heavy chain variable region (V_(H)) of“gHBC34v7” has an amino acid sequence according to SEQ ID NO: 41 and thelight chain variable region (V_(L)) of “gHBC34v7” has an amino acidsequence according to SEQ ID NO: 59.

Particularly preferably, the antibody, or the antigen binding fragmentthereof, according to the present invention is gHBC34v23, in particular,the antibody, or the antigen binding fragment thereof, according to thepresent invention is HBC34v23.

The present inventors have identified a further monoclonal antibody(mAb) according to the present invention, which is referred to herein asHBC34v23 (cf. Example 12). Based on that antibody HBC34v23, inparticular on the VH and VL genes of HBC34v23, the term “gHBC34v23”, asused herein, refer to a respective “generic” antibody, or antigenbinding fragments thereof. Namely, “gHBC34v23” refers to an antibody, orantigen binding fragment thereof, having a CDRH1 amino acid sequenceaccording to SEQ ID NO: 34, a CDRH2 amino acid sequence according to SEQID NO: 35 or 66, a CDRH3 amino acid sequence according to SEQ ID NO: 36,a CDRL1 amino acid sequence according to SEQ ID NO: 37, a CDRL2 aminoacid sequence according to SEQ ID NO: 38 or 39, and a CDRL3 amino acidsequence according to SEQ ID NO: 58. The heavy chain variable region(V_(H)) of “gHBC34v23” has an amino acid sequence according to SEQ IDNO: 41 and the light chain variable region (V_(L)) of “gHBC34v23” has anamino acid sequence according to SEQ ID NO: 59.

Particularly preferably, the antibody, or the antigen binding fragmentthereof, according to the present invention is gHBC34v31, in particular,the antibody, or the antigen binding fragment thereof, according to thepresent invention is HBC34v31.

The present inventors have identified a further monoclonal antibody(mAb) according to the present invention, which is referred to herein asHBC34v31 (cf. Example 12). Based on that antibody HBC34v31, inparticular on the VH and VL genes of HBC34v31, the term “gHBC34v31”, asused herein, refer to a respective “generic” antibody, or antigenbinding fragments thereof. Namely, “gHBC34v31” refers to an antibody, orantigen binding fragment thereof, having a CDRH1 amino acid sequenceaccording to SEQ ID NO: 34, a CDRH2 amino acid sequence according to SEQID NO: 35 or 66, a CDRH3 amino acid sequence according to SEQ ID NO: 36,a CDRL1 amino acid sequence according to SEQ ID NO: 37, a CDRL2 aminoacid sequence according to SEQ ID NO: 38 or 39, and a CDRL3 amino acidsequence according to SEQ ID NO: 40. The heavy chain variable region(V_(H)) of “gHBC34v31” has an amino acid sequence according to SEQ IDNO: 67 and the light chain variable region (V_(L)) of “gHBC34v31” has anamino acid sequence according to SEQ ID NO: 42.

Particularly preferably, the antibody, or the antigen binding fragmentthereof, according to the present invention is gHBC34v32, in particular,the antibody, or the antigen binding fragment thereof, according to thepresent invention is HBC34v32.

The present inventors have identified a further monoclonal antibody(mAb) according to the present invention, which is referred to herein asHBC34v32 (cf. Example 12). Based on that antibody HBC34v32, inparticular on the VH and VL genes of HBC34v32, the term “gHBC34v32”, asused herein, refer to a respective “generic” antibody, or antigenbinding fragments thereof. Namely, “gHBC34v32” refers to an antibody, orantigen binding fragment thereof, having a CDRH1 amino acid sequenceaccording to SEQ ID NO: 34, a CDRH2 amino acid sequence according to SEQID NO: 35 or 66, a CDRH3 amino acid sequence according to SEQ ID NO: 36,a CDRL1 amino acid sequence according to SEQ ID NO: 37, a CDRL2 aminoacid sequence according to SEQ ID NO: 38 or 39, and a CDRL3 amino acidsequence according to SEQ ID NO: 40. The heavy chain variable region(V_(H)) of “gHBC34v32” has an amino acid sequence according to SEQ IDNO: 67 and the light chain variable region (V_(L)) of “gHBC34v32” has anamino acid sequence according to SEQ ID NO: 59.

Particularly preferably, the antibody, or the antigen binding fragmentthereof, according to the present invention is gHBC34v33, in particular,the antibody, or the antigen binding fragment thereof, according to thepresent invention is HBC34v33.

The present inventors have identified a further monoclonal antibody(mAb) according to the present invention, which is referred to herein asHBC34v33 (cf. Example 12). Based on that antibody HBC34v33, inparticular on the VH and VL genes of HBC34v33, the term “gHBC34v33”, asused herein, refer to a respective “generic” antibody, or antigenbinding fragments thereof. Namely, “gHBC34v33” refers to an antibody, orantigen binding fragment thereof, having a CDRH1 amino acid sequenceaccording to SEQ ID NO: 34, a CDRH2 amino acid sequence according to SEQID NO: 35 or 66, a CDRH3 amino acid sequence according to SEQ ID NO: 36,a CDRL1 amino acid sequence according to SEQ ID NO: 37, a CDRL2 aminoacid sequence according to SEQ ID NO: 38 or 39, and a CDRL3 amino acidsequence according to SEQ ID NO: 40. The heavy chain variable region(V_(H)) of “gHBC34v33” has an amino acid sequence according to SEQ IDNO: 67 and the light chain variable region (V_(L)) of “gHBC34v33” has anamino acid sequence according to SEQ ID NO: 65.

Preferably, the antibody according to the present invention, or theantigen binding fragment thereof, is a human antibody, a monoclonalantibody, a human monoclonal antibody, a purified antibody, a singlechain antibody, Fab, Fab′, F(ab′)2, Fv or scFv.

The antibodies of the invention may thus be human antibodies, monoclonalantibodies, human monoclonal antibodies, recombinant antibodies orpurified antibodies. The invention also provides fragments of theantibodies of the invention, particularly fragments that retain theantigen-binding activity of the antibodies. Such fragments include, butare not limited to, single chain antibodies, Fab, Fab′, F(ab′)2, Fv orscFv.

Fragments of the antibodies of the invention can be obtained from theantibodies by methods that include digestion with enzymes, such aspepsin or papain, and/or by cleavage of disulfide bonds by chemicalreduction. Alternatively, fragments of the antibodies can be obtained bycloning and expression of part of the sequences of the heavy or lightchains. Antibody “fragments” include Fab, Fab′, F(ab′)2 and Fvfragments. The invention also encompasses single-chain Fv fragments(scFv) derived from the heavy and light chains of an antibody of theinvention. For example, the invention includes a scFv comprising theCDRs from an antibody of the invention. Also included are heavy or lightchain monomers and dimers, single domain heavy chain antibodies, singledomain light chain antibodies, as well as single chain antibodies, e.g.,single chain Fv in which the heavy and light chain variable domains arejoined by a peptide linker.

Antibody fragments of the invention may impart monovalent or multivalentinteractions and be contained in a variety of structures as describedabove. For instance, scFv molecules may be synthesized to create atrivalent “triabody” or a tetravalent “tetrabody”. The scFv moleculesmay include a domain of the Fc region resulting in bivalent minibodies.In addition, the sequences of the invention may be a component ofmultispecific molecules in which the sequences of the invention targetthe epitopes of the invention and other regions of the molecule bind toother targets. Exemplary molecules include, but are not limited to,bispecific Fab2, trispecific Fabs, bispecific scFv, and diabodies(Holliger and Hudson, 2005, Nature Biotechnology 9: 1126-1136).

In another aspect, the present invention provides the antibody, or theantigen binding fragment thereof, according to the present invention asdescribed herein for use as a medicament. Preferably, the antibody, orthe antigen binding fragment thereof, according to the present inventionas described herein is for use in the prophylaxis, treatment orattenuation of hepatitis B and/or hepatitis D. A detailed description ofthat aspect is provided below under “Medical treatment and uses” and inthe context of the pharmaceutical composition of the present invention.

Nucleic Acid Molecule

In another aspect, the invention also provides a nucleic acid moleculecomprising a polynucleotide encoding the antibody, or the antigenbinding fragment thereof, according to the present invention asdescribed above. Examples of nucleic acid molecules and/orpolynucleotides include, e.g., a recombinant polynucleotide, a vector,an oligonucleotide, an RNA molecule such as an rRNA, an mRNA, an miRNA,an siRNA, or a tRNA, or a DNA molecule such as a cDNA. Nucleic acidsequences encoding part or all of the light and heavy chains and CDRs ofthe antibodies of the present invention are preferred. Tables 2-8provide the SEQ ID numbers for the amino acid sequences of the CDRs andVH and VL of exemplary antibodies according to the present invention,which are preferably encoded by the polynucleotides/nucleic acidsequences as described herein. Preferably provided herein are thusnucleic acid sequences encoding part or all of the light and heavychains, in particular VH and VL sequences and CDRs of the exemplaryantibodies of the invention. Table 9 below provides the SEQ ID numbersfor the nucleic acid sequences encoding the CDRs and VH and VL anexemplified antibody according to the present invention. Due to theredundancy of the genetic code, the present invention also comprisessequence variants of these nucleic acid sequences and in particular suchsequence variants, which encode the same amino acid sequences.

A nucleic acid molecule is a molecule comprising, preferably consistingof nucleic acid components. The term nucleic acid molecule preferablyrefers to DNA or RNA molecules. In particular, it is used synonymouswith the term “polynucleotide”. Preferably, a nucleic acid molecule is apolymer comprising or consisting of nucleotide monomers which arecovalently linked to each other by phosphodiester-bonds of asugar/phosphate-backbone. The term “nucleic acid molecule” alsoencompasses modified nucleic acid molecules, such as base-modified,sugar-modified or backbone-modified etc. DNA or RNA molecules.

Table 9 shows the nucleic acid sequences of the CDR's and the heavychain variable region (VH) and the light chain variable region (VL) ofexemplary antibodies according to the present invention (“HBC34”,“HBC34v7”, “HBC34v23”, “HBC34v31”, “HBC34v32” and “HBC34v33”):

SEQ ID Name NO. Nucleic acid sequence HBC34 CDRH1 43GGACGCATCTTTAGAAGTTTTTAC CDRH2 44 ATAAACCAAGATGGAAGTGAGAAA CDRH3 45GCGGCTTGGAGCGGCAATAGTGGGGGTATGGACG TC CDRL1 46 AAATTGGGGAATAAAAAT CDRL247 GAGGTTAAA CDRL2 48 gtcatctatGAGGTTAAAtaccgcccc long CDRL3 49CAGACGTGGGACAGCACCACTGTGGTG VH 50 GAACTGCAGCTGGTGGAGTCTGGGGGAGGCTGGGTCCAGCCGGGGGGGTCCCAGAGACTGTCCT GTGCAGCCTCTGGACGCATCTTTAGAAGTTTTTACATGAGCTGGGTCCGCCAGGCCCCAGGGAAG GGGCTGGAGTGGGTGGCCACTATAAACCAAGATGGAAGTGAGAAATTATATGTGGACTCTGTGAA GGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCACTATTTCTGCAAATGAACAACCTGAGA GTCGAGGACACGGCCGTTTATTACTGCGCGGCTTGGAGCGGCAATAGTGGGGGTATGGACGTCTG GGGCCAGGGGACCACGGTCTCCGTCTCCTCA VL 51TCCTATGAGCTGACTCAGCCACCCTCAGTGTCCGTGTCCCCAGGACAGACAGTCAGCATCCCCTGCTCTGGAGATAAATTGGGGAATAAAAATGTTTGCTGGTTTCAGCATAAGCCAGGCCAGTCCCCTGTGTTGGTCATCTAT GAGGTTAAATACCGCCCCTCGGGGATTCCTGAGCGATTCTCTGGCTCCAACTCTGGGAACACAGCCACTCT GACCATCAGCGGGACCCAGGCTATGGATGAGGCTGCCTATTTCTGTCAGACGTGGGACAGCACCACTGTG GTGTTCGGCGGAGGGACCAGGCTGACCGTCCTAVH codon 70 GAACTGCAGCTGGTCGAATCAGGAGGAGGGTGGGT optimizedCCAGCCCGGAGGGAGCCAGAGACTGTCTTGTGCCG CATCAGGGAGGATCTTCAGGAGCTTCTACATGTCCTGGGTGCGCCAGGCACCAGGCAAGGGACTGGAGTG GGTCGCCACCATCAACCAGGACGGATCTGAAAAGCTGTATGTGGATAGTGTCAAAGGCCGGTTCACAATTAGCAGAGACAACGCTAAAAATTCTCTGTTTCTGCAGATGAACAATCTGCGAGTGGAGGATACCGCCGTCTACTATTGCGCCGCTTGGTCTGGCAACAGCGGCGGGATGG ATGTCTGGGGGCAGGGCACAACAGTGAGCGTCTCTTCC VL codon 71 TCATACGAACTGACTCAGCCTCCCTCCGTCTCCGTC optimizedTCACCTGGACAGACCGTCTCAATCCCCTGCTCCGGC GATAAACTGGGCAACAAGAACGTGTGCTGGTTCCAGCA CAAACCCGGACAGAGTCCTGTGCTGGTCATCTACGAGGTCAAGTATCGGCCAAGCGGCATTCCCGAAAGATTCAGCGGCTCCAACTCTGGGAATACCGCAACACTGACTATCTCTGGAACCCAGGCAATGGACGAGGCAGCTTACTTTTGCCAGACTTGGGATTCAACTACTGTCGTGTT CGGCGGCGGAACTAGACTGACTGTCCTG CRDH172 GGGAGGATCTTCAGGAGCTTCTAC codon optimized CDRH2 73ATCAACCAGGACGGATCTGAAAAG codon optimized CDRH3 74GCCGCTTGGTCTGGCAACAGCGGCGGGATGGATGT codon C optimized CDRL1 75AAACTGGGCAACAAGAAC codon optimized CDRL2 76 GAGGTCAAG codon optimizedCDRL2 long 77 GTCATCTACGAGGTCAAGTATCGGCCA codon optimized CDRL3 78CAGACTTGGGATTCAACTACTGTCGTG codon optimizedHBC34v7, HBC34v23, HBC34 v31, HBC34 v32 and HBC34 v33 CDRL1 v7 60AAGCTGGGGAACAAAAAT and CDRL1 v23 CDRL2 v7 61 GAGGTGAAA and CDRL2 v23CDRL2 long 62 GTCATCTACGAGGTGAAATATCGGCCT v7 and CDRL2 v23 long CDRL3 v763 CAGACATTCGATTCCACCACAGTGGTC and CDRL3 v23 VL v7 64TCTTACGAGCTGACACAGCCACCTAGCGTGTCCGTCTCTCCAGGACAGACCGTGTCCATCCCTTGCTCTGGC GACAAGCTGGGGAACAAAAATGTCTGTTGGTTCCAGCACAAGCCAGGGCAGAGTCCCGTGCTGGTCATCTA CGAGGTGAAATATCGGCCTTCAGGAATTCCAGAACGGTTCAGCGGATCAAACAGCGGCAATACTGCAACCC TGACAATTAGCGGGACCCAGGCCATGGACGAAGCCGCTTATTTCTGCCAGACATTCGATTCCACCACAGTG GTCTTTGGCGGGGGAACTAGGCTGACCGTGCTGHBC34 68 GAGGTGCAGCTGGTGGAATCCGGCGGGGGACTGG v31,TGCAGCCTGGCGGCTCACTGAGACTGAGCTGTGCA HBC34 v32GCTTCTGGAAGAATCTTCAGATCTTTTTACATGAG and HBC34TTGGGTGAGACAGGCTCCTGGGAAGGGACTGGAGT v33 VHGGGTCGCAAACATCAATCAGGACGGATCAGAAAAGC TGTATGTGGATAGCGTCAAAGGCAGGTTCACTATTTCCCGCGACAACGCCAAAAATTCTCTGTTTCTGCAG ATGAACAATCTGCGGGTGGAGGATACCGCTGTCTACTATTGTGCAGCCTGGTCTGGCAACAGTGGAGGCA TGGACGTGTGGGGACAGGGAACCACAGTGACAGTCAGCTCC VL v23 69 TCTTACGAGCTGACACAGCCCCCTAGCGTGTCCGTCTCTCCAGGCCAGACAGCATCCATCACTTGCTCTGGC GACAAGCTGGGGAACAAAAATGCCTGTTGGTATCAGCAGAAGCCAGGGCAGAGTCCCGTGCTGGTCATCT ACGAGGTGAAATATCGGCCTTCAGGAATTCCAGAAAGATTCAGTGGATCAAACAGCGGCAATACTGCTACCC TGACAATTAGCGGGACCCAGGCCATGGACGAAGCTGATTACTATTGCCAGACATTCGATTCCACCACAGTG GTCTTTGGCGGGGGAACTAAGCTGACCGTGCTG

Preferably, the sequence of the nucleic acid molecule according to thepresent invention comprises or consists of a nucleic acid sequenceaccording to any one of SEQ ID NOs: 43-51 or a functional sequencevariant thereof. It is also preferred that the sequence of the nucleicacid molecule according to the present invention comprises or consistsof a nucleic acid sequence according to any one of SEQ ID NOs: 43-51,60-64 and 68-78.

It is also preferred that nucleic acid sequences according to theinvention include nucleic acid sequences having at least 70%, at least75%, at least 80%, at least 85%, at least 88%, at least 90%, at least92%, at least 95%, at least 96%, at least 97%, at least 98% or at least99% identity to the nucleic acid encoding a CDR, a VH sequence and/or aVL sequence used in an (exemplary) antibody according to the presentinvention, for example to the sequences shown in Table 9. Thus a nucleicacid molecule is preferred, wherein the polynucleotide sequencecomprises or consists of a nucleic acid sequence according to any one ofSEQ ID NOs: 43-51 or a functional sequence variant thereof. A nucleicacid molecule, wherein the polynucleotide sequence comprises or consistsof a nucleic acid sequence sharing at least 80%, preferably at least85%, more preferably at least 90%, even more preferably at least 95% andparticularly preferably at least 98% or 99% sequence identity with anyof SEQ ID NOs: 43-51, 60-64 and 68-78 is also preferred. Morepreferably, the polynucleotide sequence comprises or consists of anucleic acid sequence according to any one of SEQ ID NOs: 43-51, 60-64and 68-78.

More preferably, the polynucleotide sequence comprises or consists of anucleic acid sequence sharing at least 80%, preferably at least 85%,more preferably at least 90%, even more preferably at least 95% andparticularly preferably at least 98% or 99% sequence identity with anyof SEQ ID NOs: 70-78. SEQ ID NOs: 70-78 are codon-optimized nucleic acidsequences (cf. Table 9). Particularly preferably the polynucleotidesequence comprises or consists of a nucleic acid sequence according toany one of SEQ ID NOs: 70-78.

In general, the nucleic acid molecule may be manipulated to insert,delete or alter certain nucleic acid sequences. Changes from suchmanipulation include, but are not limited to, changes to introducerestriction sites, to amend codon usage, to add or optimizetranscription and/or translation regulatory sequences, etc. It is alsopossible to change the nucleic acid to alter the encoded amino acids.For example, it may be useful to introduce one or more (e.g., 1, 2, 3,4, 5, 6, 7, 8, 9, 10, etc.) amino acid substitutions, deletions and/orinsertions into the antibody's amino acid sequence. Such point mutationscan modify effector functions, antigen-binding affinity,post-translational modifications, immunogenicity, etc., can introduceamino acids for the attachment of covalent groups (e.g., labels) or canintroduce tags (e.g., for purification purposes). Mutations can beintroduced in specific sites or can be introduced at random, followed byselection (e.g., molecular evolution). For instance, one or more nucleicacids encoding any of the CDR regions, a VH sequence and/or a VLsequence of an (exemplary) antibody of the invention can be randomly ordirectionally mutated to introduce different properties in the encodedamino acids. Such changes can be the result of an iterative processwherein initial changes are retained and new changes at other nucleotidepositions are introduced. Further, changes achieved in independent stepsmay be combined. Different properties introduced into the encoded aminoacids may include, but are not limited to, enhanced affinity.

Vector

Further included within the scope of the invention are vectors, forexample, expression vectors, comprising a nucleic acid moleculeaccording to the present invention. Preferably, a vector comprises anucleic acid molecule as described above.

The term “vector” refers to a nucleic acid molecule, preferably to arecombinant nucleic acid molecule, i.e. a nucleic acid molecule whichdoes not occur in nature. A vector in the context of the presentinvention is suitable for incorporating or harboring a desired nucleicacid sequence. Such vectors may be storage vectors, expression vectors,cloning vectors, transfer vectors etc. A storage vector is a vectorwhich allows the convenient storage of a nucleic acid molecule. Thus,the vector may comprise a sequence corresponding, e.g., to a desiredantibody or antibody fragment thereof according to the presentinvention. An expression vector may be used for production of expressionproducts such as RNA, e.g. mRNA, or peptides, polypeptides or proteins.For example, an expression vector may comprise sequences needed fortranscription of a sequence stretch of the vector, such as a promotersequence. A cloning vector is typically a vector that contains a cloningsite, which may be used to incorporate nucleic acid sequences into thevector. A cloning vector may be, e.g., a plasmid vector or abacteriophage vector. A transfer vector may be a vector which issuitable for transferring nucleic acid molecules into cells ororganisms, for example, viral vectors. A vector in the context of thepresent invention may be, e.g., an RNA vector or a DNA vector.Preferably, a vector is a DNA molecule. For example, a vector in thesense of the present application comprises a cloning site, a selectionmarker, such as an antibiotic resistance factor, and a sequence suitablefor multiplication of the vector, such as an origin of replication.Preferably, a vector in the context of the present application is aplasmid vector.

Cells

In a further aspect, the present invention also provides cell expressingthe antibody, or the antigen binding fragment thereof, according to thepresent invention; and/or comprising the vector according the presentinvention.

Examples of such cells include but are not limited to, eukaryotic cells,e.g., yeast cells, animal cells or plant cells. Preferably, the cellsare mammalian cells, more preferably a mammalian cell line. Preferredexamples include human cells, CHO cells, HEK293T cells, PER.C6 cells,NS0 cells, human liver cells, e.g. Hepa RG cells, myeloma cells orhybridoma cells.

In particular, the cell may be transfected with a vector according tothe present invention, preferably with an expression vector. The term“transfection” refers to the introduction of nucleic acid molecules,such as DNA or RNA (e.g. mRNA) molecules, into cells, preferably intoeukaryotic cells. In the context of the present invention, the term“transfection” encompasses any method known to the skilled person forintroducing nucleic acid molecules into cells, preferably intoeukaryotic cells, such as into mammalian cells. Such methods encompass,for example, electroporation, lipofection, e.g. based on cationic lipidsand/or liposomes, calcium phosphate precipitation, nanoparticle basedtransfection, virus based transfection, or transfection based oncationic polymers, such as DEAE-dextran or polyethylenimine etc.Preferably, the introduction is non-viral.

Moreover, the cells of the present invention may be transfected stablyor transiently with the vector according to the present invention, e.g.for expressing the antibody, or the antigen binding fragment thereof,according to the present invention. Preferably, the cells are stablytransfected with the vector according to the present invention encodingthe antibody, or the antigen binding fragment thereof, according to thepresent invention. Alternatively, it is also preferred that the cellsare transiently transfected with the vector according to the presentinvention encoding the antibody, or the antigen binding fragmentthereof, according to the present invention.

Optional Additional Features of the Antibodies

Antibodies of the invention may be coupled, for example, to a drug fordelivery to a treatment site or coupled to a detectable label tofacilitate imaging of a site comprising cells of interest. Methods forcoupling antibodies to drugs and detectable labels are well known in theart, as are methods for imaging using detectable labels. Labeledantibodies may be employed in a wide variety of assays, employing a widevariety of labels. Detection of the formation of an antibody-antigencomplex between an antibody of the invention and an epitope of intereston HBsAg, in particular on the antigenic loop region of HBsAg, can befacilitated by attaching a detectable substance to the antibody.Suitable detection means include the use of labels such asradionuclides, enzymes, coenzymes, fluorescers, chemiluminescers,chromogens, enzyme substrates or co-factors, enzyme inhibitors,prosthetic group complexes, free radicals, particles, dyes, and thelike. Examples of suitable enzymes include horseradish peroxidase,alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examplesof suitable prosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material is luminol; examples of bioluminescentmaterials include luciferase, luciferin, and aequorin; and examples ofsuitable radioactive material include 125I, 131I, 35S, or 3H. Suchlabeled reagents may be used in a variety of well-known assays, such asradioimmunoassays, enzyme immunoassays, e.g., ELISA, fluorescentimmunoassays, and the like. Labeled antibodies according to the presentinvention may be thus be used in such assays for example as described inU.S. Pat. Nos. 3,766,162; 3,791,932; 3,817,837; and 4,233,402.

An antibody according to the invention may be conjugated to atherapeutic moiety such as a cytotoxin, a therapeutic agent, or aradioactive metal ion or radioisotope. Examples of radioisotopesinclude, but are not limited to, I-131, I-123, I-125, Y-90, Re-188,Re-186, At-211, Cu-67, Bi-212, Bi-213, Pd-109, Tc-99, In-111, and thelike. Such antibody conjugates can be used for modifying a givenbiological response; the drug moiety is not to be construed as limitedto classical chemical therapeutic agents. For example, the drug moietymay be a protein or polypeptide possessing a desired biologicalactivity. Such proteins may include, for example, a toxin such as abrin,ricin A, pseudomonas exotoxin, or diphtheria toxin.

Techniques for conjugating such therapeutic moiety to antibodies arewell known. See, for example, Arnon et al. (1985) “Monoclonal Antibodiesfor Immunotargeting of Drugs in Cancer Therapy,” in MonoclonalAntibodies and Cancer Therapy, ed. Reisfeld et al. (Alan R. Liss, Inc.),pp. 243-256; ed. Hellstrom et al. (1987) “Antibodies for Drug Delivery,”in Controlled Drug Delivery, ed. Robinson et al. (2d ed; Marcel Dekker,Inc.), pp. 623-653; Thorpe (1985) “Antibody Carriers of Cytotoxic Agentsin Cancer Therapy: A Review,” in Monoclonal Antibodies '84: Biologicaland Clinical Applications, ed. Pinchera et al. pp. 475-506 (EditriceKurtis, Milano, Italy, 1985); “Analysis, Results, and Future Prospectiveof the Therapeutic Use of Radiolabeled Antibody in Cancer Therapy,” inMonoclonal Antibodies for Cancer Detection and Therapy, ed. Baldwin etal. (Academic Press, New York, 1985), pp. 303-316; and Thorpe et al.(1982) Immunol. Rev. 62:119-158.

Alternatively, an antibody, or antibody fragment thereof, can beconjugated to a second antibody, or antibody fragment thereof, to forman antibody heteroconjugate as described in U.S. Pat. No. 4,676,980. Inaddition, linkers may be used between the labels and the antibodies ofthe invention, e.g., as described in U.S. Pat. No. 4,831,175. Antibodiesor, antigen-binding fragments thereof may be directly labeled withradioactive iodine, indium, yttrium, or other radioactive particle knownin the art, e.g., as described in U.S. Pat. No. 5,595,721. Treatment mayconsist of a combination of treatment with conjugated and non-conjugatedantibodies administered simultaneously or subsequently e.g., asdescribed in WO00/52031; WO00/52473.

Antibodies of the invention may also be attached to a solid support.Additionally, antibodies of the invention, or functional antibodyfragments thereof, can be chemically modified by covalent conjugation toa polymer to, for example, increase their circulating half-life.Examples of polymers, and methods to attach them to peptides, are shownin U.S. Pat. Nos. 4,766,106; 4,179,337; 4,495,285 and 4,609,546. In someembodiments the polymers may be selected from polyoxyethylated polyolsand polyethylene glycol (PEG). PEG is soluble in water at roomtemperature and has the general formula: R(O—CH₂—CH₂)_(n)O—R, wherein Rcan be hydrogen, or a protective group such as an alkyl or alkanolgroup. Preferably, the protective group may have between 1 and 8carbons. For example, the protective group is methyl. The symbol n is apositive integer. In one embodiment n is between 1 and 1,000. In anotherembodiment n is between 2 and 500. Preferably, the PEG has an averagemolecular weight between 1,000 and 40,000, more preferably the PEG has amolecular weight between 2,000 and 20,000, even more preferably the PEGhas a molecular weight between 3,000 and 12,000. Furthermore, PEG mayhave at least one hydroxy group, for example the PEG may have a terminalhydroxy group. For example, it is the terminal hydroxy group which isactivated to react with a free amino group on the inhibitor. However, itwill be understood that the type and amount of the reactive groups maybe varied to achieve a covalently conjugated PEG/antibody of the presentinvention.

Water-soluble polyoxyethylated polyols are also useful in the presentinvention. They include polyoxyethylated sorbitol, polyoxyethylatedglucose, polyoxyethylated glycerol (POG), and the like. In oneembodiment, POG is used. Without being bound by any theory, because theglycerol backbone of polyoxyethylated glycerol is the same backboneoccurring naturally in, for example, animals and humans in mono-, di-,triglycerides, this branching would not necessarily be seen as a foreignagent in the body. POG may have a molecular weight in the same range asPEG. Another drug delivery system that can be used for increasingcirculatory half-life is the liposome. Methods of preparing liposomedelivery systems are known to one of skill in the art. Other drugdelivery systems are known in the art and are described in, for example,referenced in Poznansky et al. (1980) and Poznansky (1984).

Antibodies of the invention may be provided in purified form. Typically,the antibody will be present in a composition that is substantially freeof other polypeptides e.g., where less than 90% (by weight), usuallyless than 60% and more usually less than 50% of the composition is madeup of other polypeptides.

Antibodies of the invention may be immunogenic in non-human (orheterologous) hosts e.g., in mice. In particular, the antibodies mayhave an idiotope that is immunogenic in non-human hosts, but not in ahuman host. In particular, antibodies of the invention for human useinclude those that cannot be easily isolated from hosts such as mice,goats, rabbits, rats, non-primate mammals, etc. and cannot generally beobtained by humanization or from xeno-mice.

Production of Antibodies

Antibodies according to the invention can be made by any method known inthe art. For example, the general methodology for making monoclonalantibodies using hybridoma technology is well known (Kohler, G. andMilstein, C., 1975; Kozbar et al. 1983). In one embodiment, thealternative EBV immortalization method described in WO2004/076677 isused.

A preferred method is described in WO 2004/076677. In this method Bcells producing the antibody of the invention are transformed with EBVand a polyclonal B cell activator. Additional stimulants of cellulargrowth and differentiation may optionally be added during thetransformation step to further enhance the efficiency. These stimulantsmay be cytokines such as IL-2 and IL-15. In one aspect, IL-2 is addedduring the immortalization step to further improve the efficiency ofimmortalization, but its use is not essential. The immortalized B cellsproduced using these methods can then be cultured using methods known inthe art and antibodies isolated therefrom.

Another preferred method is described in WO 2010/046775. In this methodplasma cells are cultured in limited numbers, or as single plasma cellsin microwell culture plates. Antibodies can be isolated from the plasmacell cultures. Further, from the plasma cell cultures, RNA can beextracted and PCR can be performed using methods known in the art. TheVH and VL regions of the antibodies can be amplified by RT-PCR (reversetranscriptase PCR), sequenced and cloned into an expression vector thatis then transfected into HEK293T cells or other host cells. The cloningof nucleic acid in expression vectors, the transfection of host cells,the culture of the transfected host cells and the isolation of theproduced antibody can be done using any methods known to one of skill inthe art.

The antibodies may be further purified, if desired, using filtration,centrifugation and various chromatographic methods such as HPLC oraffinity chromatography. Techniques for purification of antibodies,e.g., monoclonal antibodies, including techniques for producingpharmaceutical-grade antibodies, are well known in the art.

Fragments of the antibodies of the invention can be obtained from theantibodies by methods that include digestion with enzymes, such aspepsin or papain, and/or by cleavage of disulfide bonds by chemicalreduction. Alternatively, fragments of the antibodies can be obtained bycloning and expression of part of the sequences of the heavy or lightchains. Antibody “fragments” include Fab, Fab′, F(ab′)2 and Fvfragments. The invention also encompasses single-chain Fv fragments(scFv) derived from the heavy and light chains of an antibody of theinvention. For example, the invention includes a scFv comprising theCDRs from an antibody of the invention. Also included are heavy or lightchain monomers and dimers, single domain heavy chain antibodies, singledomain light chain antibodies, as well as single chain antibodies, e.g.,single chain Fv in which the heavy and light chain variable domains arejoined by a peptide linker.

Antibody fragments of the invention may impart monovalent or multivalentinteractions and be contained in a variety of structures as describedabove. For instance, scFv molecules may be synthesized to create atrivalent “triabody” or a tetravalent “tetrabody.” The scFv moleculesmay include a domain of the Fc region resulting in bivalent minibodies.In addition, the sequences of the invention may be a component ofmultispecific molecules in which the sequences of the invention targetthe epitopes of the invention and other regions of the molecule bind toother targets. Exemplary molecules include, but are not limited to,bispecific Fab2, trispecific Fabs, bispecific scFv, and diabodies(Holliger and Hudson, 2005, Nature Biotechnology 9: 1126-1136).

Standard techniques of molecular biology may be used to prepare DNAsequences encoding the antibodies or antibody fragments of the presentinvention. Desired DNA sequences may be synthesized completely or inpart using oligonucleotide synthesis techniques. Site-directedmutagenesis and polymerase chain reaction (PCR) techniques may be usedas appropriate.

Any suitable host cell/vector system may be used for expression of theDNA sequences encoding the antibody molecules of the present inventionor fragments thereof. Bacterial, for example E. coli, and othermicrobial systems may be used, in part, for expression of antibodyfragments such as Fab and F(ab′)2 fragments, and especially Fv fragmentsand single chain antibody fragments, for example, single chain Fvs.Eukaryotic, e.g., mammalian, host cell expression systems may be usedfor production of larger antibody molecules, including complete antibodymolecules. Suitable mammalian host cells include, but are not limitedto, CHO, HEK293T, PER.C6, NS0, myeloma or hybridoma cells.

The present invention also provides a process for the production of anantibody molecule according to the present invention comprisingculturing a host cell comprising a vector encoding a nucleic acid of thepresent invention under conditions suitable for expression of proteinfrom DNA encoding the antibody molecule of the present invention, andisolating the antibody molecule.

The antibody molecule may comprise only a heavy or light chainpolypeptide, in which case only a heavy chain or light chain polypeptidecoding sequence needs to be used to transfect the host cells. Forproduction of products comprising both heavy and light chains, the cellline may be transfected with two vectors, a first vector encoding alight chain polypeptide and a second vector encoding a heavy chainpolypeptide. Alternatively, a single vector may be used, the vectorincluding sequences encoding light chain and heavy chain polypeptides.

Alternatively, antibodies according to the invention may be produced by(i) expressing a nucleic acid sequence according to the invention in ahost cell, e.g. by use of a vector according to the present invention,and (ii) isolating the expressed antibody product. Additionally, themethod may include (iii) purifying the isolated antibody. Transformed Bcells and cultured plasma cells may be screened for those producingantibodies of the desired specificity or function.

The screening step may be carried out by any immunoassay, e.g., ELISA,by staining of tissues or cells (including transfected cells), byneutralization assay or by one of a number of other methods known in theart for identifying desired specificity or function. The assay mayselect on the basis of simple recognition of one or more antigens, ormay select on the additional basis of a desired function e.g., to selectneutralizing antibodies rather than just antigen-binding antibodies, toselect antibodies that can change characteristics of targeted cells,such as their signaling cascades, their shape, their growth rate, theircapability of influencing other cells, their response to the influenceby other cells or by other reagents or by a change in conditions, theirdifferentiation status, etc.

Individual transformed B cell clones may then be produced from thepositive transformed B cell culture. The cloning step for separatingindividual clones from the mixture of positive cells may be carried outusing limiting dilution, micromanipulation, single cell deposition bycell sorting or another method known in the art.

Nucleic acid from the cultured plasma cells can be isolated, cloned andexpressed in HEK293T cells or other known host cells using methods knownin the art.

The immortalized B cell clones or the transfected host-cells of theinvention can be used in various ways e.g., as a source of monoclonalantibodies, as a source of nucleic acid (DNA or mRNA) encoding amonoclonal antibody of interest, for research, etc.

The invention also provides a composition comprising immortalized Bmemory cells or transfected host cells that produce antibodies accordingto the present invention.

The immortalized B cell clone or the cultured plasma cells of theinvention may also be used as a source of nucleic acid for the cloningof antibody genes for subsequent recombinant expression.

Expression from recombinant sources is more common for pharmaceuticalpurposes than expression from B cells or hybridomas e.g., for reasons ofstability, reproducibility, culture ease, etc.

Thus the invention also provides a method for preparing a recombinantcell, comprising the steps of: (i) obtaining one or more nucleic acids(e.g., heavy and/or light chain mRNAs) from the B cell clone or thecultured plasma cells that encodes the antibody of interest; (ii)inserting the nucleic acid into an expression vector and (iii)transfecting the vector into a host cell in order to permit expressionof the antibody of interest in that host cell.

Similarly, the invention provides a method for preparing a recombinantcell, comprising the steps of: (i) sequencing nucleic acid(s) from the Bcell clone or the cultured plasma cells that encodes the antibody ofinterest; and (ii) using the sequence information from step (i) toprepare nucleic acid(s) for insertion into a host cell in order topermit expression of the antibody of interest in that host cell. Thenucleic acid may, but need not, be manipulated between steps (i) and(ii) to introduce restriction sites, to change codon usage, and/or tooptimize transcription and/or translation regulatory sequences.

Furthermore, the invention also provides a method of preparing atransfected host cell, comprising the step of transfecting a host cellwith one or more nucleic acids that encode an antibody of interest,wherein the nucleic acids are nucleic acids that were derived from animmortalized B cell clone or a cultured plasma cell of the invention.Thus the procedures for first preparing the nucleic acid(s) and thenusing it to transfect a host cell can be performed at different times bydifferent people in different places (e.g., in different countries).

These recombinant cells of the invention can then be used for expressionand culture purposes. They are particularly useful for expression ofantibodies for large-scale pharmaceutical production. They can also beused as the active ingredient of a pharmaceutical composition. Anysuitable culture technique can be used, including but not limited tostatic culture, roller bottle culture, ascites fluid, hollow-fiber typebioreactor cartridge, modular minifermenter, stirred tank, microcarrierculture, ceramic core perfusion, etc.

Methods for obtaining and sequencing immunoglobulin genes from B cellsor plasma cells are well known in the art (e.g., see Chapter 4 of KubyImmunology, 4th edition, 2000).

The transfected host cell may be a eukaryotic cell, including yeast andanimal cells, particularly mammalian cells (e.g., CHO cells, NS0 cells,human cells such as PER.C6 or HKB-11 cells, myeloma cells, or a humanliver cell, such as Hepa RG), as well as plant cells, whereby mammaliancells are preferred. Preferred expression hosts can glycosylate theantibody of the invention, particularly with carbohydrate structuresthat are not themselves immunogenic in humans. In one embodiment thetransfected host cell may be able to grow in serum-free media. In afurther embodiment the transfected host cell may be able to grow inculture without the presence of animal-derived products. The transfectedhost cell may also be cultured to give a cell line.

The invention also provides a method for preparing one or more nucleicacid molecules (e.g., heavy and light chain genes) that encode anantibody of interest, comprising the steps of: (i) preparing animmortalized B cell clone or culturing plasma cells according to theinvention; (ii) obtaining from the B cell clone or the cultured plasmacells nucleic acid that encodes the antibody of interest. Further, theinvention provides a method for obtaining a nucleic acid sequence thatencodes an antibody of interest, comprising the steps of: (i) preparingan immortalized B cell clone or culturing plasma cells according to theinvention; (ii) sequencing nucleic acid from the B cell clone or thecultured plasma cells that encodes the antibody of interest.

The invention further provides a method of preparing nucleic acidmolecule(s) that encode an antibody of interest, comprising the step ofobtaining the nucleic acid that was obtained from a transformed B cellclone or cultured plasma cells of the invention. Thus the procedures forfirst obtaining the B cell clone or the cultured plasma cell, and thenobtaining nucleic acid(s) from the B cell clone or the cultured plasmacells can be performed at different times by different people indifferent places (e.g., in different countries).

The invention also comprises a method for preparing an antibody (e.g.,for pharmaceutical use) according to the present invention, comprisingthe steps of: (i) obtaining and/or sequencing one or more nucleic acids(e.g., heavy and light chain genes) from the selected B cell clone orthe cultured plasma cells expressing the antibody of interest; (ii)inserting the nucleic acid(s) into or using the nucleic acid(s)sequence(s) to prepare an expression vector; (iii) transfecting a hostcell that can express the antibody of interest; (iv) culturing orsub-culturing the transfected host cells under conditions where theantibody of interest is expressed; and, optionally, (v) purifying theantibody of interest.

The invention also provides a method of preparing an antibody comprisingthe steps of culturing or sub-culturing a transfected host cellpopulation, e.g. a stably transfected host cell population, underconditions where the antibody of interest is expressed and, optionally,purifying the antibody of interest, wherein said transfected host cellpopulation has been prepared by (i) providing nucleic acid(s) encoding aselected antibody of interest that is produced by a B cell clone orcultured plasma cells prepared as described above, (ii) inserting thenucleic acid(s) into an expression vector, (iii) transfecting the vectorin a host cell that can express the antibody of interest, and (iv)culturing or sub-culturing the transfected host cell comprising theinserted nucleic acids to produce the antibody of interest. Thus theprocedures for first preparing the recombinant host cell and thenculturing it to express antibody can be performed at very differenttimes by different people in different places (e.g., in differentcountries).

Pharmaceutical Compositions

The present invention also provides a pharmaceutical compositioncomprising one or more of:

-   (i) the antibody, or the antibody fragment thereof, according to the    present invention;-   (ii) the nucleic acid encoding the antibody, or antibody fragments    according to the present invention;-   (iii) the vector comprising the nucleic acid according to the    present invention; or-   (iv) the cell expressing the antibody according to the present    invention or comprising the vector according to the present    invention.

In other words, the present invention also provides a pharmaceuticalcomposition comprising the antibody, or the antigen binding fragmentthereof, according to the present invention, the nucleic acid accordingto the present invention, the vector according to the present inventionand/or the cell according to the present invention.

The pharmaceutical composition may preferably also contain apharmaceutically acceptable carrier, diluent and/or excipient. Althoughthe carrier or excipient may facilitate administration, it should notitself induce the production of antibodies harmful to the individualreceiving the composition. Nor should it be toxic. Suitable carriers maybe large, slowly metabolized macromolecules such as proteins,polypeptides, liposomes, polysaccharides, polylactic acids, polyglycolicacids, polymeric amino acids, amino acid copolymers and inactive virusparticles. In general, pharmaceutically acceptable carriers in apharmaceutical composition according to the present invention may beactive components or inactive components. Preferably, thepharmaceutically acceptable carrier in a pharmaceutical compositionaccording to the present invention is not an active component in respectto hepatitis B or D.

Pharmaceutically acceptable salts can be used, for example mineral acidsalts, such as hydrochlorides, hydrobromides, phosphates and sulphates,or salts of organic acids, such as acetates, propionates, malonates andbenzoates.

Pharmaceutically acceptable carriers in a pharmaceutical composition mayadditionally contain liquids such as water, saline, glycerol andethanol. Additionally, auxiliary substances, such as wetting oremulsifying agents or pH buffering substances, may be present in suchcompositions. Such carriers enable the pharmaceutical compositions to beformulated as tablets, pills, dragees, capsules, liquids, gels, syrups,slurries and suspensions, for ingestion by the subject.

Pharmaceutical compositions of the invention may be prepared in variousforms. For example, the compositions may be prepared as injectables,either as liquid solutions or suspensions. Solid forms suitable forsolution in, or suspension in, liquid vehicles prior to injection canalso be prepared (e.g., a lyophilized composition, similar to Synagis™and Herceptin™, for reconstitution with sterile water containing apreservative). The composition may be prepared for topicaladministration e.g., as an ointment, cream or powder. The compositionmay be prepared for oral administration e.g., as a tablet or capsule, asa spray, or as a syrup (optionally flavored). The composition may beprepared for pulmonary administration e.g., as an inhaler, using a finepowder or a spray. The composition may be prepared as a suppository orpessary. The composition may be prepared for nasal, aural or ocularadministration e.g., as drops. The composition may be in kit form,designed such that a combined composition is reconstituted just prior toadministration to a subject. For example, a lyophilized antibody may beprovided in kit form with sterile water or a sterile buffer.

It is preferred that the active ingredient in the composition is anantibody molecule, an antibody fragment or variants and derivativesthereof, in particular the active ingredient in the composition is anantibody, an antibody fragment or variants and derivatives thereof,according to the present invention. As such, it may be susceptible todegradation in the gastrointestinal tract. Thus, if the composition isto be administered by a route using the gastrointestinal tract, thecomposition may contain agents which protect the antibody fromdegradation but which release the antibody once it has been absorbedfrom the gastrointestinal tract.

A thorough discussion of pharmaceutically acceptable carriers isavailable in Gennaro (2000) Remington: The Science and Practice ofPharmacy, 20th edition, ISBN: 0683306472.

Pharmaceutical compositions of the invention generally have a pH between5.5 and 8.5, in some embodiments this may be between 6 and 8, and inother embodiments about 7. The pH may be maintained by the use of abuffer. The composition may be sterile and/or pyrogen free. Thecomposition may be isotonic with respect to humans. In one embodimentpharmaceutical compositions of the invention are supplied inhermetically-sealed containers.

Within the scope of the invention are compositions present in severalforms of administration; the forms include, but are not limited to,those forms suitable for parenteral administration, e.g., by injectionor infusion, for example by bolus injection or continuous infusion.Where the product is for injection or infusion, it may take the form ofa suspension, solution or emulsion in an oily or aqueous vehicle and itmay contain formulatory agents, such as suspending, preservative,stabilizing and/or dispersing agents. Alternatively, the antibodymolecule may be in dry form, for reconstitution before use with anappropriate sterile liquid. A vehicle is typically understood to be amaterial that is suitable for storing, transporting, and/oradministering a compound, such as a pharmaceutically active compound, inparticular the antibodies according to the present invention. Forexample, the vehicle may be a physiologically acceptable liquid, whichis suitable for storing, transporting, and/or administering apharmaceutically active compound, in particular the antibodies accordingto the present invention. Once formulated, the compositions of theinvention can be administered directly to the subject. In one embodimentthe compositions are adapted for administration to mammalian, e.g.,human subjects.

The pharmaceutical compositions of this invention may be administered byany number of routes including, but not limited to, oral, intravenous,intramuscular, intra-arterial, intramedullary, intraperitoneal,intrathecal, intraventricular, transdermal, transcutaneous, topical,subcutaneous, intranasal, enteral, sublingual, intravaginal or rectalroutes. Hyposprays may also be used to administer the pharmaceuticalcompositions of the invention. Preferably, the pharmaceuticalcomposition may be prepared for oral administration, e.g. as tablets,capsules and the like, for topical administration, or as injectable,e.g. as liquid solutions or suspensions, whereby it is particularlypreferred that the pharmaceutical composition is an injectable. Solidforms suitable for solution in, or suspension in, liquid vehicles priorto injection are also be preferred, e.g. that the pharmaceuticalcomposition is in lyophilized form.

For injection, e.g. intravenous, cutaneous or subcutaneous injection, orinjection at the site of affliction, the active ingredient willpreferably be in the form of a parenterally acceptable aqueous solutionwhich is pyrogen-free and has suitable pH, isotonicity and stability.Those of relevant skill in the art are well able to prepare suitablesolutions using, for example, isotonic vehicles such as Sodium ChlorideInjection, Ringer's Injection, Lactated Ringer's Injection.Preservatives, stabilizers, buffers, antioxidants and/or other additivesmay be included, as required. Whether it is a polypeptide, peptide, ornucleic acid molecule, other pharmaceutically useful compound accordingto the present invention that is to be given to an individual,administration is preferably in a “prophylactically effective amount” ora “therapeutically effective amount” (as the case may be), this beingsufficient to show benefit to the individual. The actual amountadministered, and rate and time-course of administration, will depend onthe nature and severity of what is being treated. For injection, thepharmaceutical composition according to the present invention may beprovided for example in a pre-filled syringe.

The inventive pharmaceutical composition as defined above may also beadministered orally in any orally acceptable dosage form including, butnot limited to, capsules, tablets, aqueous suspensions or solutions. Inthe case of tablets for oral use, carriers commonly used include lactoseand corn starch. Lubricating agents, such as magnesium stearate, arealso typically added. For oral administration in a capsule form, usefuldiluents include lactose and dried cornstarch. When aqueous suspensionsare required for oral use, the active ingredient, i.e. the inventivetransporter cargo conjugate molecule as defined above, is combined withemulsifying and suspending agents. If desired, certain sweetening,flavoring or coloring agents may also be added.

The inventive pharmaceutical composition may also be administeredtopically, especially when the target of treatment includes areas ororgans readily accessible by topical application, e.g. includingdiseases of the skin or of any other accessible epithelial tissue.Suitable topical formulations are readily prepared for each of theseareas or organs. For topical applications, the inventive pharmaceuticalcomposition may be formulated in a suitable ointment, containing theinventive pharmaceutical composition, particularly its components asdefined above, suspended or dissolved in one or more carriers. Carriersfor topical administration include, but are not limited to, mineral oil,liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene,polyoxypropylene compound, emulsifying wax and water. Alternatively, theinventive pharmaceutical composition can be formulated in a suitablelotion or cream. In the context of the present invention, suitablecarriers include, but are not limited to, mineral oil, sorbitanmonostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol,2-octyldodecanol, benzyl alcohol and water.

Dosage treatment may be a single dose schedule or a multiple doseschedule, whereby in the context of the present invention a multipledose schedule is preferred. Known antibody-based pharmaceuticals, inparticular anti-HBV based pharmaceuticals, e.g. Hepatect® CP, provideguidance relating to frequency of administration in particular inrespect to different indications, e.g., whether a pharmaceutical shouldbe delivered daily, weekly, monthly, etc. Frequency and dosage may alsodepend on the severity of symptoms.

For example, the pharmaceutical composition according to the presentinvention may be administered daily, e.g. once or several times per day,e.g. once, twice, three times or four times per day, preferably once ortwice per day, more preferable once per day, for 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 or more days, e.g.daily for 1, 2, 3, 4, 5, 6 months. Preferably, the pharmaceuticalcomposition according to the present invention may be administeredweekly, e.g. once or twice per week, for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 or more weeks, e.g. weeklyfor 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months or weekly for 2, 3,4, or 5 years. Moreover, the pharmaceutical composition according to thepresent invention may be preferably administered monthly, e.g. once permonth or, more preferably, every second month for 1, 2, 3, 4, or 5 ormore years. A preferred endpoint of administration is whenseroconversion is reached, preferably the endpoint of therapy is thepersistent disappearance of HBsAg from serum, accompanied byseroconversion to anti-HBV antibodies. It is also preferred that theadministration continues for the lifetime. In addition, also one singleadministration only is also envisaged, in particular in respect tocertain indications, e.g. for prevention of hepatitis B in case ofaccidental exposure in non-immunised subjects.

In particular, it is preferred that for a single dose, e.g. a daily,weekly or monthly dose, preferably for a weekly dose, the amount of theantibody, or the antigen binding fragment thereof, in the pharmaceuticalcomposition according to the present invention, does not exceed 1 g,preferably does not exceed 500 mg, more preferably does not exceed 250mg, even more preferably does not exceed 100 mg, and particularlypreferably does not exceed 50 mg.

Pharmaceutical compositions typically include an “effective” amount ofone or more antibodies of the invention, i.e. an amount that issufficient to treat, ameliorate, attenuate or prevent a desired diseaseor condition, or to exhibit a detectable therapeutic effect. Therapeuticeffects also include reduction or attenuation in pathogenic potency orphysical symptoms. The precise effective amount for any particularsubject will depend upon their size, weight, and health, the nature andextent of the condition, and the therapeutics or combination oftherapeutics selected for administration. The effective amount for agiven situation is determined by routine experimentation and is withinthe judgment of a clinician. For purposes of the present invention, aneffective dose will generally be from about 0.005 to about 100 mg/kg,preferably from about 0.0075 to about 50 mg/kg, more preferably fromabout 0.01 to about 10 mg/kg, and even more preferably from about 0.02to about 5 mg/kg, of the antibody of the present invention (e.g. amountof the antibody in the pharmaceutical composition) in relation to thebodyweight (e.g., in kg) of the individual to which it is administered.

For example, in the context of liver transplantation, e.g. due tohepatitis B induced liver failure, the amount of the antibody, or theantigen binding fragment thereof, in the pharmaceutical compositionaccording to the present invention, may preferably not exceed 50 mg,more preferably not more than 10 mg, for a single dose on the day oftransplantation, peri-operatively then not more than 10-50 mg,preferably not more than 2-10 mg, per day for seven days and not morethan 10-50 mg, preferably not more than 2-10 mg, per single doseadministered every 1-3 months to maintain anti-HBs serum levels about100 IU/L.

For the treatment of chronic hepatitis B, for example, the antibody, orthe antigen binding fragment thereof, or the pharmaceutical compositionaccording to the present invention, is preferably administeredsubcutaneously, with a single dose of up to 500 mg, preferably of up to250 mg, more preferably of up to 100 mg of the antibody according to thepresent invention. Such a single dose may be administered daily, weeklyor monthly as described above.

Moreover, the pharmaceutical composition according to the presentinvention may also comprise an additional active component, which may bea further antibody or a component, which is not an antibody. Theadditional active component is preferably selected from polymeraseinhibitors, interferons and/or checkpoint inhibitors. Preferredpolymerase inhibitors include Lamivudine, Adefovir, Entecavir,Telbivudine and Tenofovir. Polymerase inhibitors suppress reversetranscription and synthesis of the DNA-plus strand. Polymeraseinhibitors do not prevent viral spread, formation of cccDNA and does notaffect HBsAg release. Interferons include IFNalpha and IFNbeta, wherebyIFNbeta is preferred. Preferred checkpoint inhibitors are directed to ablockade of PD-1/PD-L1 and/or of CTLA4 and, thus, include anti-PD-1antibodies, anti-PD-L1 antibodies and anti-CTLA4 antibodies. Thepharmaceutical composition according to the present invention maycomprise one or more of the additional active components.

The antibody, or the antigen binding fragment, according to the presentinvention can be present either in the same pharmaceutical compositionas the additional active component or, preferably, the antibody, or theantigen binding fragment, according to the present invention iscomprised by a first pharmaceutical composition and the additionalactive component is comprised by a second pharmaceutical compositiondifferent from the first pharmaceutical composition. Accordingly, ifmore than one additional active component is envisaged, each additionalactive component and the antibody, or the antigen binding fragment,according to the present invention is preferably comprised by adifferent pharmaceutical composition. Such different pharmaceuticalcompositions may be administered either combined/simultaneously or atseparate times or at separate locations (e.g. separate parts of thebody).

Preferably, antibody, or the antigen binding fragment, according to thepresent invention and the additional active component provide anadditive therapeutic effect or, preferably, a synergistic therapeuticeffect. The term “synergy” is used to describe a combined effect of twoor more active agents that is greater than the sum of the individualeffects of each respective active agent. Thus, where the combined effectof two or more agents results in “synergistic inhibition” of an activityor process, it is intended that the inhibition of the activity orprocess is greater than the sum of the inhibitory effects of eachrespective active agent. The term “synergistic therapeutic effect”refers to a therapeutic effect observed with a combination of two ormore therapies wherein the therapeutic effect (as measured by any of anumber of parameters) is greater than the sum of the individualtherapeutic effects observed with the respective individual therapies.

A pharmaceutical composition comprising the antibody according to gHCB34or an antigen binding fragment thereof, and a pharmaceuticallyacceptable carrier is preferred.

In one embodiment, a composition of the invention may include antibodiesof the invention, wherein the antibodies may make up at least 50% byweight (e.g., 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% ormore) of the total protein in the composition. In such a composition,the antibodies are preferably in purified form.

The present invention also provides a method of preparing apharmaceutical composition comprising the steps of (i) preparing anantibody of the invention; and (ii) admixing the purified antibody withone or more pharmaceutically-acceptable carriers.

In another embodiment, a method of preparing a pharmaceuticalcomposition comprises the step of: admixing an antibody with one or morepharmaceutically-acceptable carriers, wherein the antibody is amonoclonal antibody that was obtained from a transformed B cell or acultured plasma cell of the invention.

As an alternative to delivering antibodies or B cells for therapeuticpurposes, it is possible to deliver nucleic acid (typically DNA) thatencodes the monoclonal antibody (or active fragment thereof) of interestderived from the B cell or the cultured plasma cells to a subject, suchthat the nucleic acid can be expressed in the subject in situ to providea desired therapeutic effect. Suitable gene therapy and nucleic aciddelivery vectors are known in the art.

Pharmaceutical compositions may include an antimicrobial, particularlyif packaged in a multiple dose format. They may comprise detergent e.g.,a Tween (polysorbate), such as Tween 80. Detergents are generallypresent at low levels e.g., less than 0.01%. Compositions may alsoinclude sodium salts (e.g., sodium chloride) to give tonicity. Forexample, a concentration of 10±2 mg/ml NaCl is typical.

Further, pharmaceutical compositions may comprise a sugar alcohol (e.g.,mannitol) or a disaccharide (e.g., sucrose or trehalose) e.g., at around15-30 mg/ml (e.g., 25 mg/ml), particularly if they are to be lyophilizedor if they include material which has been reconstituted fromlyophilized material. The pH of a composition for lyophilization may beadjusted to between 5 and 8, or between 5.5 and 7, or around 6.1 priorto lyophilization.

The compositions of the invention may also comprise one or moreimmunoregulatory agents. In one embodiment, one or more of theimmunoregulatory agents include(s) an adjuvant.

Medical Treatments and Uses

In a further aspect, the present invention provides the use of anantibody, or an antigen binding fragment thereof, according to thepresent invention, the nucleic acid according to the present invention,the vector according to the present invention, the cell according to thepresent invention or the pharmaceutical composition according to thepresent invention in (i) prophylaxis, treatment or attenuation ofhepatitis B and/or hepatitis D; or in (ii) diagnosis of hepatitis Band/or hepatitis D.

Within the scope of the invention are several forms and routes ofadministration of the antibody, or the antigen binding fragment thereof,the nucleic acid, the vector, the cell or the pharmaceuticalcomposition, as described above in respect to the pharmaceuticalcomposition. This applies also in the context of the use of theantibody, or the antigen binding fragment thereof, the nucleic acid, thevector, and the cell as described herein, in particular regardingpreferred forms and routes of administration.

Methods of diagnosis may include contacting an antibody or an antibodyfragment with a sample. Such samples may be isolated from a subject, forexample an isolated tissue sample taken from, for example, nasalpassages, sinus cavities, salivary glands, lung, liver, pancreas,kidney, ear, eye, placenta, alimentary tract, heart, ovaries, pituitary,adrenals, thyroid, brain, skin or blood, preferably serum. The methodsof diagnosis may also include the detection of an antigen/antibodycomplex, in particular following the contacting of an antibody or anantibody fragment with a sample. Such a detection step is typicallyperformed at the bench, i.e. without any contact to the human or animalbody. Examples of detection methods are well-known to the person skilledin the art and include, e.g., ELISA (enzyme-linked immunosorbent assay).

The invention also provides the use of (i) an antibody, an antibodyfragment, or variants and derivatives thereof according to theinvention, (ii) an immortalized B cell clone according to the invention,(iii) a nucleic acid or a vector according to the present invention or(iv) a pharmaceutical composition of the invention in (a) themanufacture of a medicament for the prevention, treatment or attenuationof hepatitis B and/or hepatitis D or for (b) diagnosis of hepatitis Band/or hepatitis D.

The invention also provides the antibody, or an antigen binding fragmentthereof, according to the present invention, the nucleic acid accordingto the present invention, the vector according to the present invention,the cell according to the present invention or the pharmaceuticalcomposition according to the present invention for use as a medicament,in particular for the prevention or treatment of hepatitis B and/orhepatitis D. It also provides the use of an antibody of the invention inthe manufacture of a medicament for treatment of a subject and/ordiagnosis in a subject. It also provides a method for treating asubject, comprising the step of administering to the subject acomposition of the invention. In some embodiments the subject may be ahuman. One way of checking efficacy of therapeutic treatment involvesmonitoring disease symptoms after administration of the composition ofthe invention. Treatment can be a single dose schedule or a multipledose schedule.

In one embodiment, an antibody, antibody fragment, immortalized B cellclone, or pharmaceutical composition according to the invention isadministered to a subject in need of such treatment. Such a subjectincludes, but is not limited to, one who is particularly at risk of orsusceptible to hepatitis B and/or hepatitis D.

Antibodies, or antigen binding fragments thereof, according to thepresent invention may also be used in a kit for the diagnosis ofhepatitis B and/or hepatitis D. Further, the epitope in the antigenicloop region of HBsAg, which is capable of binding an antibody of theinvention as described herein may be used in a kit for monitoring theefficacy of application procedures by detecting the presence ordetermining the titer of protective anti-HBV antibodies.

Preferably, the antibody, or an antigen binding fragment thereof,according to the present invention, the nucleic acid according to thepresent invention, the vector according to the present invention, thecell according to the present invention or the pharmaceuticalcomposition according to the present invention is used in treatment orattenuation of chronic hepatitis B.

Interestingly, the antibody according to the present invention (i)potently neutralizes HBV infection, (ii) binds to L-HBsAg (the large HBVenvelope protein, which is present in infectious HBV particles), therebypreventing spreading of HBV, (iii) binds to S-HBsAg, thereby promotingclearance of subviral particles (SVP) and (iv) can induceseroconversion, i.e. an active immune response to the virus.

Preferably, the antibody, or an antigen binding fragment thereof,according to the present invention, the nucleic acid according to thepresent invention, the vector according to the present invention, thecell according to the present invention or the pharmaceuticalcomposition according to the present invention is used in prevention ofhepatitis B (re-)infection after liver transplantation in particular forhepatitis B induced liver failure.

To this end, preferably a high dose may be administered to the patientreceiving a liver transplantation on the day of transplantation anddaily doses are given peri-operatively for about a week. Thereafter,preferably further doses may be given every 1-3 months to maintainanti-HBV antibody serum levels above 100 IU/ml.

In another preferred embodiment the antibody, or an antigen bindingfragment thereof, according to the present invention, the nucleic acidaccording to the present invention, the vector according to the presentinvention, the cell according to the present invention or thepharmaceutical composition according to the present invention is used inprevention/prophylaxis of hepatitis B in non-immunized subjects. This isfor example in case of (an assumed) accidental exposure to HBV(post-exposure prophylaxis). The term “non-immunized subjects” includessubjects, who never received a vaccination and are, thus, not immunized,and subjects, who did not show an immune response (no measurableanti-hepatitis B antibodies) after vaccination. In particular in thelatter group, the antibody, or an antigen binding fragment thereof,according to the present invention, the nucleic acid according to thepresent invention, the vector according to the present invention, thecell according to the present invention or the pharmaceuticalcomposition according to the present invention is used in (continuous)prevention of hepatitis B, i.e. in contrast to “post-exposureprophylaxis” (continuous) prevention is preferably for such subjects,who did not show an immune response (no measurable anti-hepatitis Bantibodies) after vaccination and for whom continuous prevention isnecessary.

Preferably, the antibody, or an antigen binding fragment thereof,according to the present invention, the nucleic acid according to thepresent invention, the vector according to the present invention, thecell according to the present invention or the pharmaceuticalcomposition according to the present invention is used in prophylaxis ofhepatitis B in haemodialysed patients.

Preferably, the antibody, or an antigen binding fragment thereof,according to the present invention, the nucleic acid according to thepresent invention, the vector according to the present invention, thecell according to the present invention or the pharmaceuticalcomposition according to the present invention is used in prevention ofhepatitis B in the newborn. Thereby, in particular newborns of hepatitisB virus carrier-mothers/non-immunized mothers are preferred. Moreover,it is preferred that the antibody, or an antigen binding fragmentthereof, according to the present invention, the nucleic acid accordingto the present invention, the vector according to the present invention,the cell according to the present invention or the pharmaceuticalcomposition according to the present invention is administered at birthor as soon as possible after birth. Preferably, the administration maybe repeated until seroconversion following vaccination.

Preferably, the antibody, or an antigen binding fragment thereof,according to the present invention, the nucleic acid according to thepresent invention, the vector according to the present invention, thecell according to the present invention or the pharmaceuticalcomposition according to the present invention is used in treatment orattenuation of hepatitis D, preferably of hepatitis B and hepatitis D,which is in particular a hepatitis B and hepatitis D comorbidity.Interestingly, the antibody according to the present invention does notonly potently neutralize hepatitis B virus, but also hepatitis deltavirus. Therefore, the antibody according to the present invention mayprovide a first treatment of hepatitis D.

Combination Therapy

The administration of the antibody, or an antigen binding fragmentthereof, according to the present invention, the nucleic acid accordingto the present invention, the vector according to the present invention,the cell according to the present invention or the pharmaceuticalcomposition according to the present invention in the methods and usesaccording to the invention can be carried out alone or in combinationwith a co-agent (also referred to as “additional active component”herein) useful for treating and/or stabilizing the disease or disorderto be treated or repressed.

The invention encompasses the administration of the antibody, or anantigen binding fragment thereof, according to the present invention,the nucleic acid according to the present invention, the vectoraccording to the present invention, the cell according to the presentinvention or the pharmaceutical composition according to the presentinvention, wherein it is administered to a subject prior to,simultaneously or sequentially with other therapeutic regimens orco-agents useful for treating, and/or preventing hepatitis B. Saidantibody, nucleic acid, vector, cell or pharmaceutical composition, thatis administered simultaneously with said co-agents can be administeredin the same or different composition(s) and by the same or differentroute(s) of administration.

Said other therapeutic regimens or co-agents may be selected from thegroup consisting of polymerase inhibitors, interferons and/or acheckpoint inhibitors.

Thus, in another aspect of the present invention the antibody, or anantigen binding fragment thereof, according to the present invention,the nucleic acid according to the present invention, the vectoraccording to the present invention, the cell according to the presentinvention or the pharmaceutical composition according to the presentinvention is administered in combination with a polymerase inhibitor, aninterferon and/or a checkpoint inhibitor for the above described(medical) uses.

Preferred polymerase inhibitors include Lamivudine, Adefovir, Entecavir,Telbivudine and Tenofovir. Lamivudine is the most preferred polymeraseinhibitor. Polymerase inhibitors suppress revers transcription andsynthesis of the DNA-plus strand. Polymerase inhibitors do not preventviral spread, formation of cccDNA and does not affect HBsAg release.

Interferons include IFNalpha and IFNbeta, whereby IFNbeta is preferred.

Preferred checkpoint inhibitors are directed to a blockade of PD-1/PD-L1and/or of CTLA4 and, thus, include anti-PD-1 antibodies, anti-PD-L1antibodies and anti-CTLA4 antibodies.

Thus, the pharmaceutical composition according to the present inventionmay comprise one or more of the additional active components.

Further preferred co-agent (additional active components) to beadministered in combination with the antibody, or an antigen bindingfragment thereof, according to the present invention, the nucleic acidaccording to the present invention, the vector according to the presentinvention, the cell according to the present invention or thepharmaceutical composition according to the present invention includeLTβR agonists.

Preferably, the antibody, or the antigen binding fragment thereof,according to the present invention, the nucleic acid according to thepresent invention, the vector according to the present invention, thecell according to the present invention or the pharmaceuticalcomposition according to the present invention is administered incombination with a polymerase inhibitor. This applies in particular fora use of such a combination in prophylaxis, treatment or attenuation ofhepatitis B and/or hepatitis D. In this context, preferred polymeraseinhibitors include Lamivudine, Adefovir, Entecavir, Telbivudine andTenofovir. The most preferred polymerase inhibitor is lamivudine.

Preferably, the antibody, or the antigen binding fragment thereof, thenucleic acid, the vector, the cell or the pharmaceutical composition isadministered via the same or a distinct route of administration as thepolymerase inhibitor, the interferon and/or the checkpoint inhibitor.

In a further aspect the present invention thus also provides acombination of

-   -   (i) the antibody, or the antigen binding fragment thereof,        according to the present invention, the nucleic acid according        to the present invention, the vector according to the present        invention, the cell according to the present invention or the        pharmaceutical composition according to the present invention;        and    -   (ii) a polymerase inhibitor, an interferon and/or a checkpoint        inhibitor.

Such a combination is preferably used in prophylaxis, treatment orattenuation of hepatitis B and/or hepatitis D, in particular intreatment or attenuation of chronic hepatitis B and/or chronic hepatitisD. More preferably, the combination is used in HBV mono-infectedpatients or in HBV/HDV co-infected patients.

Such a combination preferably accelerates HBsAg clearance.

In view thereof, the present invention also provides a kit comprising

-   -   (i) the antibody, or the antigen binding fragment thereof,        according to the present invention, the nucleic acid according        to the present invention, the vector according to the present        invention, the cell according to the present invention or the        pharmaceutical composition according to the present invention;        and    -   (ii) a polymerase inhibitor, an interferon and/or a checkpoint        inhibitor.

In addition, the kit may comprise means for administration of theantibody, or an antigen binding fragment thereof, according to thepresent invention, the nucleic acid according to the present invention,the vector according to the present invention, the cell according to thepresent invention or the pharmaceutical composition according to thepresent invention, such as a syringe or a vessel and/or a leaflet, forexample with instructions on the use of the antibody, or the antigenbinding fragment thereof, according to the present invention, thenucleic acid according to the present invention, the vector according tothe present invention, the cell according to the present invention orthe pharmaceutical composition according to the present invention and/orthe polymerase inhibitor, the interferon and/or the checkpointinhibitor.

The antibody, or the antigen binding fragment, according to the presentinvention can be present either in the same pharmaceutical compositionas the additional active component (co-agent) or, preferably, theantibody, or the antigen binding fragment, according to the presentinvention is comprised by a first pharmaceutical composition and theadditional active component (co-agent) is comprised by a secondpharmaceutical composition different from the first pharmaceuticalcomposition. Accordingly, if more than one additional active component(co-agent) is envisaged, each additional active component (co-agent) andthe antibody, or the antigen binding fragment, according to the presentinvention is preferably comprised by a different pharmaceuticalcomposition. Such different pharmaceutical compositions may beadministered either combined/simultaneously or at separate times or atseparate locations (e.g. separate parts of the body).

Preferably, antibody, or the antigen binding fragment, according to thepresent invention and the additional active component (co-agent) providean additive therapeutic effect or, preferably, a synergistic therapeuticeffect. As described above, the term “synergy” is used to describe acombined effect of two or more active agents that is greater than thesum of the individual effects of each respective active agent. Thus,where the combined effect of two or more agents results in “synergisticinhibition” of an activity or process, it is intended that theinhibition of the activity or process is greater than the sum of theinhibitory effects of each respective active agent. The term“synergistic therapeutic effect” refers to a therapeutic effect observedwith a combination of two or more therapies wherein the therapeuticeffect (as measured by any of a number of parameters) is greater thanthe sum of the individual therapeutic effects observed with therespective individual therapies.

Uses and Methods

In another aspect the present invention provides the use of theantibody, or an antigen binding fragment thereof, according to thepresent invention, the nucleic acid according to the present invention,the vector according to the present invention, the cell according to thepresent invention or the pharmaceutical composition according to thepresent invention for monitoring the quality of anti-hepatitis-B oranti-hepatitis-D vaccines by checking that the antigen of said vaccinecontains the specific epitope in the correct conformation.

Moreover, the present invention also provides the use of the antibody,or an antigen binding fragment thereof, according to the presentinvention, the nucleic acid according to the present invention, thevector according to the present invention, the cell according to thepresent invention or the pharmaceutical composition according to thepresent invention in diagnosis of hepatitis B and/or hepatitis D.

In addition also the use of the antibody, or an antigen binding fragmentthereof, according to the present invention, the nucleic acid accordingto the present invention, the vector according to the present invention,the cell according to the present invention or the pharmaceuticalcomposition according to the present invention in determining whether anisolated blood sample is infected with hepatitis B virus and/orhepatitis delta virus is provided.

As described above, methods of diagnosis may include contacting anantibody or an antibody fragment with a sample. Such samples may beisolated from a subject, for example an isolated tissue sample takenfrom, for example, nasal passages, sinus cavities, salivary glands,lung, liver, pancreas, kidney, ear, eye, placenta, alimentary tract,heart, ovaries, pituitary, adrenals, thyroid, brain, skin or blood,preferably serum. The methods of diagnosis may also include thedetection of an antigen/antibody complex, in particular following thecontacting of an antibody or an antibody fragment with a sample. Such adetection step is typically performed at the bench, i.e. without anycontact to the human or animal body. Examples of detection methods arewell-known to the person skilled in the art and include, e.g., ELISA(enzyme-linked immunosorbent assay).

The present invention also provides a method of preventing and/ortreating hepatitis B and/or hepatitis D in a subject, wherein the methodcomprises administering to a subject in need thereof the antibody, or anantigen binding fragment thereof, according to the present invention,the nucleic acid according to the present invention, the vectoraccording to the present invention, the cell according to the presentinvention or the pharmaceutical composition according to the presentinvention.

The present invention also provides a method of treating a subject whohas received a liver transplant comprising administering to the subjectwho has received the liver transplant, a therapeutically effectiveamount of the antibody, or an antigen binding fragment thereof,according to the present invention, the nucleic acid according to thepresent invention, the vector according to the present invention, thecell according to the present invention or the pharmaceuticalcomposition according to the present invention.

In the above methods a subject suffering from chronic hepatitis B ispreferred.

Moreover, the previously described details, in particular in the contextof the pharmaceutical composition and the medical uses, also apply forthe methods described herein. For example, in the methods describedabove the antibody, or an antigen binding fragment thereof, according tothe present invention, the nucleic acid according to the presentinvention, the vector according to the present invention, the cellaccording to the present invention or the pharmaceutical compositionaccording to the present invention is preferably administered incombination with a polymerase inhibitor, an interferon and/or acheckpoint inhibitor as described herein.

BRIEF DESCRIPTION OF THE FIGURES

In the following a brief description of the appended figures will begiven. The figures are intended to illustrate the present invention inmore detail. However, they are not intended to limit the subject matterof the invention in any way.

FIG. 1 shows for Example 1 the binding of HBC34 monoclonal antibody tothree different HBsAg serotypes (adw, ady, and ayw) as measured byELISA.

FIG. 2 shows for Example 2 the ability of various anti-HB antibodies,namely HBV immunoglobulins (HBIG), HBC34, and further monoclonalantibodies against PreS1 (18/7) or HBsAg to neutralize HBV infection ofHepaRG cell in vitro. Each antibody was tested at three differentconcentrations, namely 5 μg/ml, 0.5 μg/ml and 0.05 μg/ml, except forHBIG, which was tested at 5000 μg/ml, 500 μg/ml and 50 μg/ml.

FIG. 3 shows for Example 2 the staining of HBcAg in HepaRG cellsinfected in the presence of three different concentrations (5 μg/ml, 0.5μg/ml or 0.05 μg/ml) of HBC34 monoclonal antibody and, as a reference,the nuclear staining.

FIG. 4 shows for Example 2 the neutralization activity of differentconcentrations of HBC34 on infectious HDV. At a concentration of 0.12μg/ml HBC34 no HDV-positive cells were detectable, indicating potentneutralization of HDV. HBIG, in contrast, did not neutralize HDV (testedin 1:1000 dilution, i.e. 50 μg/ml).

FIG. 5 shows for Example 3 the amino acid sequences of the antigenicloop of HBsAg of the 10 HBV genotypes A, B. C. D, E, F, G, H, I and J.Those sequences comprise the epitope recognized by the HBC34 antibody(highlighted in grey). The sequence on the top (HBV-D J02203) was usedto design the peptide library in Example 5, FIG. 7.

FIG. 6 shows for Example 3 the binding, as determined bycytofluorimetric analysis, of the human monoclonal antibody HBC34 and acontrol antibody (both at 5 μg/ml) to permeabilized Hep2 cellstransiently transfected with plasmids expressing the different HBsAggenotypes A, B, C, D, E, F, G, H, I and J as indicated in the Figure.

FIG. 7 shows for Example 4 the amino acid sequences of the antigenicloop of the 19 HBsAg mutants tested. Circled are the residues of theHBsAg mutants that were weakly (dotted circle) or not bound by HBC34antibody.

FIGS. 8A and 8B show for Example 4 the binding of the human monoclonalantibody HBC34 and two other HBsAg-specific antibodies (Ab5 and Ab6) alltested at 5 μg/ml on Hep2 cells transfected with plasmids expressing thedifferent HBsAg genotype D mutants as indicated in the Figure (“WT”:HBsAg Genotype D, Genbank accession no. FJ899792).

FIG. 9 shows for Example 5 the binding of HBC34 to a library of 650linear and looped peptides as determined using the Pepscan technology aswell as the sequences of the four peptides bound by HBC34. Residuesindicate as 1 are cysteines that were introduced to allow the chemicallinking to scaffolds in order to reconstruct conformational epitopes. Ifother cysteines besides the newly introduced cysteines are present, theyare replaced by alanine (underlined alanine residues).

FIG. 10 shows for Example 5 a western blot staining by Ab4 and HBC34 onHBV viral particles under reducing conditions. Ab4 is a comparativeantibody, which is also reactive against the antigenic loop.

FIG. 11 shows for Example 6 the levels of HBV viremia in humanizeduPA/SCID mice inoculated with 5×10⁷ copies of HBV genome equivalents(genotype D), which received from three weeks post-infection treatmentwith either HBC34 (at 1 mg/kg administered i.p. twice per week), acontrol antibody (control AB) or entecavir (ETV; administered orally at1 μg/ml) for 6 weeks. In the spreading phase of HBV infection (weeks 3to week 6 p.i. (post infection)) viremia increased >2 log in the groupwhich received the control antibody, while HBV titers decreased in micetreated with HBC34 or entecavir.

FIG. 12 shows for Example 6 staining of hepatocytes for the presence ofHBsAg (intrahepatical analysis) in mice of Example 6 at the end of theexperiment (week 9). Nearly all hepatocytes stained HBsAg positive inmice which received the control antibody, while spreading wasefficiently blocked by both, treatment with entecavir and treatment withHBC34 (ca. 1-5% HBsAg-positive cells).

FIG. 13 shows for Example 6 cccDNA measurements, which did not differsignificantly between mice that were sacrificed 3 weeks post HBVinfection (“week hbv”; i.e. no treatment) and mice that were treatedfrom week 3 to week 9 post-infection with HBC34 or entecavir. Incontrast, the estimated amount of cccDNA/cell increased up to 2 logs inthe group receiving the control antibody when sacrificed 9 weekspost-infection.

FIG. 14 shows for Example 6 levels of circulating HBsAg at baseline(BL), week 3 of treatment (week 6 post-infection) and week 6 oftreatment (week 9 post-infection). Levels of circulating HBsAgdecreased >1 log (and below the limit of detection) in mice receivingHBC34, but not in mice treated with entecavir, while HBsAg levelsincreased >2 logs (reaching levels of 5000-10000 IU/ml) in the controlgroup.

FIG. 15 shows for Example 7 HBV titer (left panel) and levels ofcirculating HBsAg (right panel) in a chronic hepatitis B setting atbaseline (BL), week 3 of treatment (week 15 post-infection, “week 3”)and week 6 of treatment (week 18 post-infection, “week 6”). HBV titerand levels of circulating HBsAg were decreased in mice receiving HBC34at week 3 and week 6 of treatment. Individual curves representindividual animals. Control antibody: dotted lines, HBC34: continuouslines.

FIG. 16 shows for Example 8 HBV titer (left panel) and HDV titer (rightpanel) in uPA/SCID mice repopulated with primary human hepatocytes andco-infected with a patient-derived serum containing HDV-RNA and HBV-DNA.Five weeks after infection treatment with HBC34 or a control antibody(“control”) was started. HBV titer (left panel) and HDV titer (rightpanel) are shown at baseline (week3, week5BL), at week 3 of treatment(week 8 post-infection—“week 8”) and at week 6 of treatment (week 11post-infection—“week 11”). Individual curves represent individualanimals. Control antibody: dotted lines, HBC34: continuous lines.

FIG. 17 (part 1) shows for Example 9 a schematics of the antigenic loopof HBV-s-Antigen, the epitope of HBC34 is highlighted in grey. To mapthe epitope of HBC34 a library of 1520 different peptides was tested asdetermined using the Pepscan technology. FIG. 17 (part 2-5) show forExample 9 the magnitude of binding (ELISA intensities) of HBC34 to 16different sets of peptides. HBC34 is binding to a conformational epitopeas demonstrated by the binding to peptides of sets 13-16 composed ofcombinatorial CLIPS constructs, representing two parts of adiscontinuous epitope (part 2) and to peptides of sets 9-12 composed oflooped peptides (part 3). No binding of HBC34 is observed with sets oflinear peptides 1-4 (part 4) and 5-8 (part 5) further supporting thenotion that HBC34 binds to a discontinuous conformational epitope.

FIG. 18 shows for Example 9 the binding of HBC34 to a peptide librarycomposed of 812 discontinuous or looped T3 CLIPS peptides. In order tofine tune the epitope mapping described in FIG. 17, 3 sets of peptides(dubbed RN1, RN2 and RNs) were generated based on the previous sets(FIG. 17) by means of full substitution analysis. FIG. 18 shows bindingof HBC34 to sets 1-3 where residues at the boxed positions weresubstituted with by one of 13 amino acids selected from seriesAEFGHKLPQRSVY_, where “_” stands for residue deletion. The originalresidue at the permutated position is indicated on the left of eachpanel as well as either (i) in the horizontally boxed sequence (when theoriginal amino acid is part of the permutation series) or (ii) below thesequence (when the original amino acid is not part of the permutationseries).

FIG. 19 shows for Example 10 the effect of a combination therapy withHBC34 (I mg/kg i.p twice a week) and Lamivudine (supplemented at 0.4mg/ml in drinking water) in reducing the levels of HBV viremia (panel A)and circulating HBV-s-Antigen (panel B) in humanized uPA/SCID miceinoculated with 2×10⁹ copies of HBV genome equivalents (genotype D),which received for 4 weeks starting from 8 weeks post-infection,treatment with either a control antibody (control), HBC34 alone (HBC34),lamividune alone (Lamivudine) or a combination treatment (HBC34 andlamivudine). Combination therapy caused higher reduction of viremiacompared with either drugs alone.

FIG. 20 shows for Examples 11 and 12 the alignments of the two sets ofVH (panel A) and VL (panel B) sequences generated to obtain the antibodyvariants 1-32. CDRs (defined according to IMGT) are highlighted in grey.

FIG. 21 shows for Example 11 the binding of 18 engineered HBC34 variants(obtained by combining the mutated VH and VL sequences as indicated incolumns 2 and 3) to HBsAg (adw subtype) as determined by ELISA. Incolumns 4, loss of binding is indicated with “−”, strongly reducedbinding is indicated with “+/−”, reduced binding is indicated with“+/˜”, binding similar or equal to the original antibody is indicatedwith “+”.

FIG. 22 shows for Example 11 binding of 8 different engineered variantsof HBC34 to HBsAg (adw) as determine in direct antigen-based ELISAassay. These 8 variants were select among the 18 HBC34 mutants describedin FIG. 21; in order to better characterize the affinity for HBsAg the 8antibodies were titrated and compared with the parental antibodysequence.

FIG. 23 shows for Example 11 the summary of the characteristics of the 8antibodies described in FIG. 22. EC50 were determined by fitting thecurves in FIG. 22 using Graphpad prism. Productivity was determined by(ELISA) quantification of secreted IgG in the supernatant of a 300 mltransfection of 293 Expi cells with each of the 8 variants as well asthe parental antibody.

FIG. 24 shows for Examples 11 and 12 a table summarizing thecharacteristics of 15 variants, among which are 12 additional engineeredvariants of HBC34, designed based on the results of the previous set byintroducing additional mutations in the frameworks (panel A). Bindingcurves were obtained by titrating the antibodies in antigen-based ELISAassay and EC50 were calculated by fitting the curves with Graphpadprism. Productivities were calculated based on quantification of the IgGsecreted in the superntatant of a 30 ml transfection of 293 Expi cellswith each of the 15 variants and the parental antibody. Fold-changeswere plotted and are shown in panel B.

FIG. 25 shows for Examples 11 and 12 the binding, as determined bycytofluorimetric analysis, of HBC34 and variants 6, 7, 19.23 and 24. Allantibodies were titrated starting from 5 μg/ml and bound topermeabilized Hep2 cells transiently transfected with plasmidsexpressing the different HBsAg genotypes A, B, C, D, E, F, G, H, I andJ.

EXAMPLES

In the following, particular examples illustrating various embodimentsand aspects of the invention are presented. However, the presentinvention shall not to be limited in scope by the specific embodimentsdescribed herein. The following preparations and examples are given toenable those skilled in the art to more clearly understand and topractice the present invention. The present invention, however, is notlimited in scope by the exemplified embodiments, which are intended asillustrations of single aspects of the invention only, and methods whichare functionally equivalent are within the scope of the invention.Indeed, various modifications of the invention in addition to thosedescribed herein will become readily apparent to those skilled in theart from the foregoing description, accompanying figures and theexamples below. All such modifications fall within the scope of theappended claims.

Example 1: Identification and Characterization of Human MonoclonalAntibody HBC34

A human monoclonal antibody was isolated in a similar manner asdescribed in Traggiai E. et al., 2004, Nat Med 10(8): 871-5 from a humanpatient. The antibody was characterized by determining the nucleotideand amino acid sequences of its variable regions (Tables 2 and 3) andthe complementarity determining regions (CRDs) therein and termed“HBC34”. Accordingly, HBC34 is an IgG1-type fully human monoclonalantibody having the CDR, VH and VL sequences as shown above in Tables 2and 3.

Next, it was determined to which of the three HBsAg serotypes adw, ady,and ayw the human monoclonal antibody HBC34 binds to. Interestingly,HBC34 binds with high affinity to three HBsAg serotypes (adw, ady andayw) with similar and low EC50 values, as measured by ELISA (FIG. 1).

Protective titers of HBV antibodies are expressed in International Units(IU) which allows standardization over different assays. In 1977, anInternational Reference Preparation for anti-HBs immunoglobulin (W1042)was established. The plasma used in the preparation of this standard wasderived from individuals who had been naturally infected with hepatitisB virus (Barker, L. F., D. Lorenz, S. C. Rastogi, J. S. Finlayson, andE. B. Seligmann. 1977. Study of a proposed international referencepreparation for antihepatitis B immunoglobulin. WHO Expert Committee onBiological Standardization technical report series. WHO Expert Committeeon Biological Standardisation 29th Report BS 77.1 164. Geneva,Switzerland, World Health Organization, 1977; World Health Organization:Anti-hepatitis B immunoglobulin. WHO Tech Rep Ser 1978; 626:18). Theactivity of HBC34, as measured diagnostically with an immunoassay(Abbott Architect diagnostic immunoassay), is 5000 IU/mg. As acomparison the activity of HBIG is 1 IU/mg.

Example 2: Antibody HBC34 Potently Neutralizes Infectious HBV and HDV

The first object of Example 2 was to determine whether HBC34 neutralizesinfectious HBV and to compare the neutralization activity of HBC34 tothat of other anti-HB antibodies. To this end, differentiated HepaRGcells were incubated with a fixed amount of HBV in the presence orabsence of antibodies (HBC34, 18/7, Ab2, Ab3 and HBIG) in mediumsupplemented with 4% PEG 8000 (Sigma-Aldrich) for 16 hours at 37° C. Atthe end of the incubation, the cells were washed and further cultivated.Medium was changed every 3 days. Infection was detected by measuring inenzyme-linked immunosorbent assay (ELISA) the levels of hepatitis Bsurface antigen (HBsAg) and hepatitis B e antigen (HBeAg) secreted intothe culture supernatant from day 7 to 11 post-infection and by detectingHBcAg staining in an immunofluorescence assay.

As shown in FIGS. 2 and 3, HBC34 neutralized completely HBV infectionwhen tested at 5 and 0.5 μg/ml, whereas comparative human monoclonalanti-HB antibodies Ab2 and Ab3, which are also binding to HBsAg, did notresult in complete neutralization. This indicates that not allantibodies binding to HBsAg are able to neutralize HBV infection (e.g.Ab2 and Ab3). Of note, HBIG neutralized HBV infection only when testedat 5000 and 500 μg/ml, i.e. with a 1000 fold lower potency as comparedto HBC34. 18/7 is a murine monoclonal antibody against the pre-S1 regionof HBsAg.

The second object of Example 2 was to determine the neutralizingactivity of HBC34 against HDV on differentiated HepaRg cells. Sera fromHDV carriers were used as HDV infection moculum. Delta antigenimmunofluorescence staining was used as a readout. As shown in FIG. 4,HBC34 completely blocked HDV infection when tested at 0.12 μg/ml. As acomparison, HBIG were also tested and were ineffective (tested at1/1000, i.e. 50 μg/ml).

Example 3: Antibody HBC34 Recognizes all 10 HBV Genotypes a, B, C, D, E,F, G, H, I, and J

HBC34 was tested for its ability to recognize the 10 HBV genotypes A, B,C, D, E, F, G, H, I, and J (as shown in FIG. 5) by flow cytometryanalysis. In particular, human epithelial cells (Hep2 cells) weretransfected with plasmids expressing each of the HBsAg of the 10 HBVgenotypes A, B, C, D, E, F, G, H, I, and J (as shown in FIG. 5). Humanmonoclonal antibody HBC34 (5 μg/ml) and a control antibody (5 μg/ml)were used for staining of transiently transfected permeabilized cells.Two days after transfection, Hep2 cells were collected, fixed andpermeabilized with saponin for immunostaining with HBC34 or a controlAb. Binding of antibodies to transfected cells was analysed using aBecton Dickinson FACSCanto2 (BD Biosciences) with FlowJo software(TreeStar). As shown in FIG. 6 HBC34 recognized all 10 HBV HBsAggenotypes with similar patterns of staining.

Example 4: Antibody HBC34 Recognizes all Functional HBsAg Mutants

HBC34 was tested for its ability to bind to the 19 different HBsAggenotype D mutants (based on HBsAg Genotype D, Genbank accession no.FJ899792, as shown in FIG. 7) HBsAg Y100C/P120T, HBsAg P120T, HBsAgP120T/S143L, HBsAg C121S, HBsAg R122D, HBsAg R122I, HBsAg T123N, HBsAgT123N/C124R, HBsAg Q129H, HBsAg Q129L, HBsAg M133H, HBsAg M133L, HBsAgM133T, HBsAg K141E, HBsAg P142S, HBsAg S143K, HBsAg D144A, HBsAg G145Rand HBsAg N146A (see SEQ ID NO's 16-33 for the amino acid sequences ofthe antigenic loop regions of those mutants) by flow cytometry analysis.In particular, human epithelial cells (Hep2 cells) were transfected withplasmids expressing the different HBsAg mutants and analyzed as inExample 3. 5 μg/ml of human monoclonal antibody HBC34 and two otherHBsAg-specific antibodies (Ab5 and Ab6) were used for testing thebinding of HBC34 to the transfected Hep2 cells.

As shown in FIGS. 8A-8B, HBC34 was found to bind to 18 of the 19 HBsAgmutants. HBC34 binding, but not Ab5 and Ab6 binding, was completelyabolished only in the mutant HBsAg T123N/C124R, i.e. when residues 123and 124 were both mutated. Of note, the mutation of these two residues(i.e. T123 and C124) into alanine was shown to be associated with a lossof HBV infectivity, which is most likely due to the loss of thedisulphide bridge formed by C124 that could result in a conformationalchange in the antigenic loop (Salisse J. and Sureau C., 2009, Journal ofVirology 83: 9321-9328). Thus, human monoclonal antibody HBC34 binds to18 HBsAg mutants.

Example 5: Antibody HBC34 Binds to a Conserved Conformational Epitope inthe Antigenic Loop

The epitope recognized by HBC34 was identified by using a library of 650linear and looped peptides (“CLIPS Discontinuous Epitope Mapping”technology from Pepscan, Lelystad, The Netherlands) designed to coverthe entire antigenic loop region of the HBsAg. The linear and CLIPSpeptides are synthesized based on standard Fmoc-chemistry. The loopedpeptides are synthesized on chemical scaffolds in order to reconstructconformational epitopes, using the Chemically Linked Peptides onScaffolds, CLIPS, technology as described in Timmerman et al., 2007,Journal of Molecular Recognition 20: 283-99. For example the singlelooped peptides are synthesized containing two cysteines and the size ofthe loop is varied by introducing the cysteine residues at variablespacing. If other cysteines besides the newly introduced cysteines arepresent, they are replaced by alanine. The side-chains of the multiplecysteines in the peptides are coupled to CLIPS templates by reactingonto credit-card format polypropylene PEPSCAN cards (455 peptideformats/card) with a 0.5 mM solution of CLIPS template such as 1,3-bis(bromomethyl)benzene in ammonium bicarbonate. The binding of antibody toeach peptide is tested in a PEPSCAN-based ELISA. The 455-well creditcard format polypropylene cards containing the covalently linkedpeptides are incubated with the test antibody (at 1 μg/ml) in blockingsolution. After washing the peroxidase substrate2,2′-azino-di-3-ehylbenzthiazoline sulfonate (ABTS) and 2 μl of 3% H₂O₂are added. After one hour, the color development is measured with acharge coupled device (CCD)-camera and an image processing system. Theraw data are optical values and range from 0 to 3000 (a log scalesimilar to I to 3 of a standard 96-well plate ELISA reader).

As shown in FIG. 9, HBC34 was found to recognize a double looped peptidehaving an amino acid sequence according to SE ID NO 52:

XGSSTTSTGPCRTCMTXPSDGNATAIPIPSSWX

wherein the residues coded as X were substituted with Cysteines and theunderlined residues were substituted from C to A (SEQ ID NO 52).

Three additional peptides were recognized with a lower signal:

(a) a linear 15-mer peptide having an amino acid sequence according toSEQ ID NO 53:

(SEQ ID NO 53) TSTGPCRTCMTTAQG,

(b) another linear peptide having an amino acid sequence according toSEQ ID NO 54:

(SEQ ID NO 54) GMLPVCPLIPGSSTTSTGPCRTCMTT,

and (c) a double looped peptide having an amino acid sequence accordingto SEQ ID NO 55:

XSMYPSASATKPSDGNXTGPCRTCMTTAQGTSX

-   -   wherein the residues coded as X were substituted with the        Cysteines and the underlined residues were substituted from C to        A (SEQ ID NO 55).

This analysis indicated that the core epitope of HBC34 is formed by aconformational epitope formed by an amino acid sequence according to SEQID NO: 56:

PCRTCMTTAQG

(SEQ ID NO 56; amino acids 120-130 of the S domain of HBsAg (HBV-DJ02203).

Moreover, as shown in FIG. 10 the human monoclonal antibody HBC34 doesnot react at all in a western blot on HBV viral particles under reducingconditions.

These results confirm that the epitope of HBsAg, to which HBC34 bindsto, is a conformational epitope.

These results are consistent with what observed in Example 4 where HBC34binding was lost in the presence of the T123N/C124R mutations.

The region of HBsAg, which comprises the conformational epitope, towhich HBC34 binds to, is polymorphic in the different HBV genotypes. Inthe following generic sequence of the epitope region of HBsAg theresidues mutated in the different genotypes are indicated with an X:

PCX₁TCX₂X₃X₄AQG,

wherein X₁ is preferably R or K,

-   -   X₂ is preferably M or T,    -   X₃ is preferably T or I, and    -   X₄ is preferably T, P or L

(SEQ ID NO: 57).

Moreover, an additional comparison of the above sequence to the 18 HBsAgmutants, to which the human monoclonal antibody HBC34 binds to,indicates that HBC34 binds to an epitope formed by an amino acidsequence according to SEQ ID NO: 2:

X₁X₂X₃TCX₄X₅X₆AX₇G

wherein X₁ is P, T or S,

-   -   X₂ is C or S,    -   X₃ is R, K, D or I,    -   X₄ is M or T,    -   X₅ is T, A or I,    -   X₆ is T, P or L, and    -   X₇ is Q, H or L.

Example 6: Administration of HBC34 Starting 3 Weeks Post HBV InfectionPrevents Viral Spreading in Humanized uPA Mice

The object of Example 6 was to investigate whether human monoclonalantibody HBC34 is able to prevent spreading of HBV. In other words, itwas the aim to investigate the capacity of the entry inhibitor HBC34antibody to inhibit infection of the human hepatocytes in vivo bytreating mice after the initial infection establishment. To this end,naïve uPA/SCID mice repopulated with primary human hepatocytes (seePetersen et al. Nature Biotechnology, 2008, 26:335-341) were used. Thesemice were engrafted with cryopreserved human hepatocytes by intrasplenicinjection. Eight weeks later successful repopulation of humanhepatocytes in the host liver was determined measuring human serumalbumin, which is exclusively expressed by transplanted humanhepatocytes. Mice with appropriate human albumin levels were inoculatedi.p. with 5×10⁷ copies of HBV genome equivalents (genotype D, HBeAgpositive) to permit viral entry. Three weeks after infection, thetreatment protocol started whereby mice received either HBC34 treatment(at 1 mg/kg administered i.p. twice per week), a control antibody orentecavir (ETV; administered orally at 1 μg/ml in water, BaracludeSolution, Bristol-Myers Squibb) for 6 weeks. Liver specimens removed atsacrifice were snap-frozen in liquid nitrogen for immunofluorescenceanalysis.

HBV DNA was extracted from serum samples using the QiAmp MinElute Virusspin kit (Qiagen, Hilden, Germany). HBV-specific primers andhybridization probes were used to determine HBV DNA viremia and cccDNAloads quantitatively as described previously (Volz T et al.,Gastroenterology 2007; 133: 843-852). DNA and RNA were extracted fromliver specimens using the Master Pure DNA purification kit (Epicentre,Biozym, Germany) and RNeasy RNA purification kit (Qiagen, Hilden,Germany). Intrahepatic HBV DNA values were normalized for cellular DNAcontents using the beta-globin gene kit (Roche DNA control Kit; RocheDiagnostics). Levels of rcDNA were estimated by subtracting cccDNAamounts from total HBV DNA. Viral RNAs and genomic RNAs were reversetranscribed using oligo-dT primers and the Transcriptor Kit (RocheApplied Science) and quantified by using primers specific for totalviral RNAs. HBV RNA levels were normalized to human specific GAPDH RNA.HBsAg quantification from blood samples was performed using the AbbottArchitect platform (quant. HBsAg kit, Abbott, Ireland, DiagnosticDivision), as recommended by the manufacturer. Cryostat sections ofchimeric mouse livers were immunostained using humanspecificcytokeratin-18 monoclonal (Dako, Glostrup, Denmark) to stain humanhepatocytes. For the detection of the HBV core antigen (HBcAg), thepolyclonal rabbit anti-HBcAg was used. Specific signals were visualizedby employing the Alexa-labeled secondary antibodies (Invitrogen,Darmstadt, Germany) or TSA-Fluorescein (HBcAg) System (Perkin Elmer,Jugesheim, Germany), while nuclear staining was obtained with Hoechst33342 (Invitrogen). Stained sections were analyzed by fluorescencemicroscope.

The median baseline level of HBV DNA at the beginning of the treatmentwas 2×10⁶ DNA copies/ml. In the spreading phase of HBV infection (weeks3 to week 6 p.i. (post infection)) viremia increased >2 log in the groupwhich received the control antibody, while HBV titers decreased in micetreated with HBC34 or entecavir (FIG. 11).

Mice were also analyzed intrahepatically at the end of the experiment(i.e. week 9) by staining hepatocytes for the presence of HBcAg. Nearlyall hepatocytes stained HBcAg positive in mice which received thecontrol antibody, while spreading was efficiently blocked by both,treatment with entecavir and treatment with HBC34 (ca. 1-5%HBcAg-positive cells). These results indicate that HBC34 can efficientlyblock viral spreading during the ramp-up phase of HBV infection (FIG.12).

In line with the histological and serological data, cccDNA measurementsshowed that intrahepatic cccDNA loads did not differ significantlybetween mice that were sacrificed 3 weeks post HBV infection and micethat were treated from week 3 to week 9 post-infection. In comparison,the estimated amount of cccDNA/cell increased up to 2 logs in thecontrol group sacrificed 9 weeks post-infection, suggesting that newlyformed rcDNAs could not be efficiently converted into cccDNA in treatedmice (FIG. 13). The same tendency was also found by measuring otherintrahepatic viral parameters, such as the levels of relaxed circularDNA (rcDNA) and HBV RNA transcripts.

Moreover, levels of blood circulating HBsAg were measured at baseline(BL), week 3 of treatment (week 6 post-infection) and week 6 oftreatment (week 9 post-infection). It is of note that the levels ofcirculating HBsAg decreased >1 log (and below the limit of detection) inmice receiving HBC34, but not in mice treated with entecavir, whileHBsAg levels increased >2 logs (reaching levels of 5000-10000 IU/ml) inthe control group (FIG. 14). The measurement of HBsAg was not influencedby the presence of the HBC34 antibody as determined in a spike-inexperiment where the addition of HBC34 antibody to HBsAg positive mousesera did not alter the expected measurement using the Abbott Architectdiagnostic immunoassay.

These results indicate that HBC34 can block HBV viral spread and promotethe clearance of HBsAg.

Example 7: Administration of HBC34 in Chronically HBV Infected HumanizeduPA Mice Promoted HBV and HBsAg Clearance

To mimic the hepatitis B chronic setting, naïve humanized uPA/SCID micewere infected with HBV and after 12 weeks post infection, a median levelof HBV DNA of 2×10⁹ copies/ml and a level of HBsAg of 10000 IU/ml wasreached. These levels are as high as the levels that are commonlyobserved in human patients with chronic HBV infection.

Thereafter, the mice were treated starting from week 12 post-infectioneither with HBC34 or with a control antibody for 6 weeks (I mg/kg i.p.twice per week). As shown in FIG. 15 HBV titer and levels of bloodcirculating HBsAg were decreased in mice receiving HBC34 for 3 weeks(week 15 post-infection) and 6 weeks (week 18 post-infection). Thus,HBC34 promoted a clear reduction of both HBV viremia and HBsAg levelsafter 6 weeks of treatment. HBeAg and human albumin levels were notaltered in HBC34-treated mice, which indicates the absence of livertoxicity.

Example 8: Administration of HBC34 Blocks HDV Infection In Vivo

Naïve humanized uPA/SCID mice were co-infected with a patient-derivedserum containing HDV-RNA and HBV-DNA. Five weeks after infection (whenHBV titers reached levels between 10⁷ to 10⁹, and HDV RNA reached levelsbetween 10³ to 10⁶, copies/ml) mice were treated with HBC34 or a controlantibody for 6 weeks (1 mg/kg i.p. twice per week).

HBV DNA viremia was measured as described in Examples 6 and 7. HDVviremia was determined via reverse transcription of viral RNA (extractedfrom serum samples using the QiAmp MinElute Virus Spin Kit, Qiagen,Venlo, Netherlands) and quantitative RT-PCR using the ABI Fast 1-StepVirus Master (Applied Biosystems, Carlsbad, USA), HDV specific primersand probes on a ABI Viia7 (Applied Biosystems, Carlsbad, USA).

As shown in FIG. 16, HBC34 efficiently blocked HDV viral spread both 3weeks and 6 weeks after treatment (weeks 8 and 11, respectively).Similarly to what was observed in HBV chronically infected mice, HBC34promoted a HBV viral DNA titers reduction of 2 logs (FIG. 13).

Example 9: Fine Epitope Mapping of the HBC34 Discontinuous Epitope

In order to further refine the epitope recognized by HBC34 antibodydescribed in Example 5 a new library of 1520 peptides composed of 16different sets was generated:

-   -   Set 1 (dubbed LIN15): Linear 15-mer peptides derived from the        target sequence (SEQ ID NO: 5:        QGMLPVCPLIPGSSTTSTGPCRTCMTTAQGTSMYPSCCCTKPSDGNCTCIP        IPSSWAFGKFLWEWASARFSW; J02203 (D, ayw3)) with an offset of one        residue. Native Cys residues are protected by an acetamidomethyl        group (also referred to as “Acm”; denoted as “2” in the        respective amino acid sequences).    -   Set 2 (dubbed LIN22): Linear 22-mer peptides derived from the        target sequence (SEQ ID NO: 5:        QGMLPVCPLIPGSSTTSTGPCRTCMTTAQGTSMYPSCCCTKPSDGNCTCIP    -   IPSSWAFGKFLWEWASARFSW; J02203 (D, ayw3)) with an offset of one        residue. Native Cys residues are protected by Acm (denoted “2”).    -   Set 3 (dubbed LIN30): Linear 30-mer peptides derived from the        target sequence (SEQ ID NO: 5:        QGMLPVCPLIPGSSTTSTGPCRTCMTTAQGTSMYPSCCCTKPSDGNCTCIP    -   IPSSWAFGKFLWEWASARFSW; J02203 (D, ayw3)) with an offset of one        residue. Native Cys residues are protected by Acm (denoted “2”).    -   Set 4 (dubbed LIN15.AA): Peptides of set 1, but with residues on        positions 9 and 10 replaced by Ala. When a native Ala occurred        on either position, it was replaced by Gly.    -   Set 5 (dubbed LIN22.AA): Peptides of set 2, but with residues on        positions 12 and 13 replaced by Ala. When a native Ala occurred        on either position, it was replaced by Gly.    -   Set 6 (dubbed LIN30.AA): Peptides of set 3, but with residues on        positions 16 and 17 replaced by Ala. When a native Ala occurred        on either position, it was replaced by Gly.    -   Set 7 (dubbed CYS.A): Combinatorial peptides of length 27. On        positions 1-11 and 17-27 are linear sequences, which contain        pairing Cys residues. These 11-mer sequences joined via “GGSGG”        (SEQ ID NO: 79) linker. Cys residues, which do not participate        in disulfide bridge formation are protected by Acm (denoted        “2”).    -   Set 8 (dubbed as CYS.B): Linear 22-mer sequences, which contain        two Cys forming a disulfide bridge. Cys residues, which do not        participate in disulfide bridge formation are protected by Acm        (denoted “2”).    -   Set 9 (dubbed LOOP12): Constrained peptides of length 12. On        positions 2-11 are 10-mer sequences derived from the target        sequence of Antigenic Loop of HBV-S-Ag. On positions 1 and 12        are Cys residues, which are joined by mP2 CLIPS. Native Cys        residues are protected by Acm (denoted “2”).    -   Set 10 (dubbed as LOOP15): Constrained peptides of length 15. On        positions 2-14 are 13-mer sequences derived from the target        sequence of Antigenic Loop of HBV-S-Ag. On positions 1 and 15        are Cys residues, which are joined by mP2 CLIPS. Native Cys        residues are protected by Acm (denoted “2”).    -   Set 11 (dubbed LOOP21): Constrained peptides of length 21. On        positions 2-20 are 19-mer sequences derived from the target        sequence of Antigenic Loop of HBV-S-Ag. On positions 1 and 21        are Cys residues, which are joined by mP2 CLIPS. Native Cys        residues are protected by Acm (denoted “2”).    -   Set 12 (dubbed LOOP31): Constrained peptides of length 31. On        positions 2-30 are 29-mer sequences derived from the target        sequence of Antigenic Loop of HBV-S-Ag. On positions 1 and 31        are Cys residues, which are joined by mP2 CLIPS. Native Cys        residues are protected by Acm (denoted “2”).    -   Set 13 (dubbed MAT.A): Combinatorial peptides of length 25. On        positions 2-12 and 14-24 are 11-mer peptides derived from the        target sequence of Antigenic Loop of HBV-S-Ag. On positions 1,        13 and 25 are Cys residues, which are joined by T3 CLIPS. Native        Cys residues are protected by Acm (denoted “2”).    -   Set 14 (dubbed MAT.B): Combinatorial peptides of length 28. On        positions 2-12 and 14-27 are 11-mer and 14-mer peptides        respectively. On positions 1, 13 and 28 are Cys residues, which        are joined by T3 CLIPS. Native Cys residues are protected by Acm        (denoted “2”).    -   Set 15 (dubbed MAT.C): Combinatorial peptides of length 28. On        positions 2-15 and 17-27 are 14-mer and 11-mer peptides        respectively. On positions 1, 16 and 28 are Cys residues, which        are joined by T3 CLIPS. Native Cys residues are protected by Acm        (denoted “2”).    -   Set 16 (dubbed MAT.D): Combinatorial peptides of length 31. On        positions 2-15 and 17-30 are 14-mer peptides derived from the        target sequence of Antigenic Loop of HBV-S-Ag. On positions 1,        16 and 31 are Cys residues, which are joined by T3 CLIPS. Native        Cys residues are protected by Acm (denoted “2”).

When tested under high stringency conditions antibody HBC34 did not bindany peptide present on the arrays. When tested under low stringencyconditions (5 μg/ml in 0.1% Pepscan buffer and preconditioningcontaining a combination of horse serum and ovalbumin) the antibodybound constrained and combinatorial peptides—binding to peptides fromset 14 and set 16 was somewhat lower as compared to set 13 and set 15.No binding was recorded on linear epitope mimics. Data are shown in FIG.17. These data show that antibody HBC34 recognizes a conformationaldiscontinuous epitope composed of peptide stretches ₁₈TGPCRTC₂₄ (SEQ IDNO: 80) and ₄₅GNCTCIP₅₁ (SEQ ID NO: 81), where peptide stretch₁₈TGPCRTC₂₄ (SEQ ID NO: 80) is the dominant part of the epitope (FIG.17).

To fine map the epitope of antibody HBC34 by means of full substitutionanalysis based on the results described above, 812 discontinuous orlooped T3 CLIPS peptides composed of three different set weresynthesized:

-   -   Set 1 (dubbed RN1; Discontinuous T3 CLIPS): Epitope mutant        series derived from the discontinuous mimic        C2IPIPSSWAFGCSTTSTGP2RT2C (SEQ ID NO: 82). For each position of        this sequence, a substitution analysis was performed. In other        words, for each position of the peptide sequence, variants were        made in which the original amino acid at such position was        replaced by one of the 13 amino acids selected from the group        consisting of alanine (A), glutamic acid (E), phenylalanine (F),        glycine (G), histidine (H), lysine (K), leucine (L), proline        (P), glutamine (Q), arginine (R), serine (S), valine (V),        tyrosine (Y), and “ ”; where “_” stands for residue deletion.        Native Cys residues are protected by Acm (denoted “2”).    -   Set 2 (dubbed RN2; Discontinuous T3 CLIPS): Epitope mutant        series derived from the discontinuous mimic        CGN2T2IPIPSSWAFCSTTSTGP2RT2C_(SEQ ID NO: 83). For each position        of this sequence, a substitution analysis was performed in the        same manner as for Set 1 (i.e., GN2T residues were not mutated).        Native Cys residues are protected by Acm (denoted “2”).    -   Set 3 (dubbed RN3; Loop T3 CLIPS): Epitope mutant series derived        from the looped mimic CGGGCSTTSTGP2RT2C_(SEQ ID NO: 84). For        positions 6-16 of this sequence, a substitution analysis was        performed in the same manner as for set 1. Native Cys residues        are protected by Acm (denoted “2”).

HBC34 antibody was tested in the PEPSCAN-based ELISA at 20 μg/ml on thepeptide array pre-conditioned with 0.1% SQ (Pepscan buffer containing0.1% of a combination of horse serum and ovalbumin). The peptide arrayswere incubated with HBC34 antibody solution (overnight at 4° C.). Afterwashing, the peptide arrays were incubated with a 1/1000 dilution of anappropriate antibody peroxidase conjugate (SBA) for one hour at 25° C.After washing, the peroxidase substrate2,2′-azino-di-3-ethylbenzthiazoline sulfonate (ABTS) and 20 μl/ml of 3percent H₂O₂ were added. After one hour, the color development wasmeasured. The color development was quantified with a charge coupleddevice (CCD)—camera and an image processing system.

As expected, when tested under low stringency conditions antibody HBC34bound peptides from all sets. Results of the experiment show thatresidues ₁₂₀PCR₁₂₂ and C₁₂₄ are critical for the binding, while onlycertain replacements of residues I150, I152, W156 and F158 notablydecrease the binding (FIG. 18). Moreover, data recorded on all threearrays coherently have shown that replacements of any residue withinregion ₁₁₄STTSTGPCRTC₁₂₄ (SEQ ID NO: 85) with E would decrease thebinding, while inverse replacements with R or Y increase binding.However, the same was not observed for region ₁₄₅GNCTCIPIPSSWAFC₁₅₉,(SEQ ID NO: 86).

Taken together these results suggest that antibody HBC34 recognizes adiscontinuous epitope with residues ₁₂₀PCR₁₂₂ and C₁₂₄ being crucial forthe binding. Presence of residues ₁₄₅GNCTCIPIPSSWAF₁₅₈ (SEQ ID NO: 87)was shown to provide structural context for establishing and stabilizingepitope-paratope interactions. This conclusion arose from theobservation that discontinuous epitope mimics (when ₁₄₅GNCTCIPIPSSWAF₁₅₈(SEQ ID NO: 87) and ₁₁₄STTSTGPCRTC₁₂₄ (SEQ ID NO: 85) are present in onemimic) are more tolerant to the replacements than sequence₁₁₄STTSTGPCRTC₁₂₄ (SEQ ID NO: 85) alone (set 3, RN3). Additionally, P151which fixes the torsion angles thereby providing conformational rigiditywas shown to impact the binding especially when replaced by G known forinversed properties. Replacement G145P similarly impacts binding ofHBC34. It was repeatedly observed that R/Y replacements improve thebinding to any position within motif ₁₁₄STTSTGPCRTC₁₂₄ (SEQ ID NO: 85),but not within motif ₁₄₅GNCTCIPIPSSWAF₁₅₈ (SEQ ID NO: 87), while Ereplacements decrease binding. This observation may suggest thatresidues ₁₁₄STTSTGPCRTC₁₂₄ (SEQ ID NO: 85) bind to a negatively chargedparatope within the antibody HBC34 (or close to a cluster of negativecharges) and improvement of the binding as well as the decreased resultfrom electrostatic forces and rather characterize the paratope featuresthan those of the epitope.

Example 10: Increased Reduction of HBsAg in Chronically HBV InfectedHumanized uPA Mice Treated with a Combination of HBC34 and Lamivudine

In a further study, the efficacy of a combination therapy including theantibody HBC34 was investigated. For combination with HBC34, apolymerase inhibitor, namely lamivudine, was selected.

To mimic the chronic hepatitis B setting, naïve humanized uPA/SCID micewere infected with HBV and after 8 weeks post infection, a median levelof HBV DNA of 2×10⁹ copies/ml and a level of HBsAg of 9000 IU/ml wasreached. These levels are as high as the levels that are commonlyobserved in human patients with chronic HBV infection.

Thereafter, mice were treated starting from week 8 post-infection eitherwith antibody HBC34 alone, with the polymerase inhibitors lamivudinealone, with a combination of HBC34 and lamivudine, or with a controlantibody for 4 weeks (HBC34 at 1 mg/kg i.p. twice per week; lamivudinesupplemented in drinking water at 0.4 mg/ml).

HBV viremia and HBsAG levels in serum were assessed in treatment week 0(before treatment), treatment week 2, treatment week 4, and treatmentweek 6, or in treatment week 0 (before treatment), treatment week 3 andtreatment week 6. Results are shown in FIGS. 19 A (HBV viremia) and B(HBsAG).

As shown in FIG. 19 treatment with HBC34, lamivudine or both drugs incombination caused mean 0.7 log, 1.3 log and 2.4 log reduction ofviremia (A), respectively. Notably, HBsAg (B) dropped 1.3 log (meanBL=15,600 IU/ml) in mice receiving HBC34 alone and 2.6 log (meanBL=2,600 IU/ml) in the combination group, whereas no significant HBsAgreduction (0.2 log; mean BL=9,000 IU/ml) was detected in mice treatedwith lamivudine alone.

In summary, the combination of HBC34 and lamivudine clearly achieved thestrongest effect. Interestingly, such a strong effect of the combinationof HBC34 and lamivudine was observed, even if lamivudine alone was noteffective. In view thereof, the observed strong effect of thecombination of HBC34 and lamivudine is clearly an unexpected synergisticeffect.

In summary, the surprisingly strong HBsAg reduction achieved incombination therapy proves that HBC34 antibody can be used, e.g. inchronic settings, in combination with polymerase inhibitors toaccelerate HBsAg clearance both in HBV mono-infected and HBV/HDVco-infected patients.

Example 11: Sequence Engineering of HBC34 Antibody: CDR3 of VH and VL

A first series of HBC34 mutants was generated with mutations in the CDR3of VH and VL by mutating (i) residue W107 of the VH CDR3 into either Aor F, (ii) residue M115 of the VH CDR3 into I or L, and/or (iii) residueW107 of the VL CDR3 into either A or F. A total of 18 HBC34 variantswere produced by combining the un-mutated VH or VL of HBC34 (hereafterreferred as WT, wild type or parental antibody) with differentcombinations of VH and VL mutants as illustrated in FIG. 20 and FIG. 21.

The produced HBC34 antibody variants were tested by ELISA for binding toHBsAg adw antigen, similarly as in Example 1. Results are shown in FIG.21.

Of note, the mutation of W107 of the VH CDR3 into A (in HBC34-V5,HBC34-V, HBC34-V9, HBC34-V10, HBC34-V12 and HBC34-V17 variants, FIG. 21)completely abolished HBC34 binding to HBsAg. This indicates that W107 isa key residue in the HBC34 paratope for antigen recognition. Themutation of W107 of the VH CDR3 into F (an amino acid with similararomatic characteristic as W) partially affected HBC34 binding(HBC34-V1), indicating that W cannot be mutated without compromisingHBC34 binding affinity to HBsAg.

The mutation of M115 of the VH CDR3 into L did not affect HBC34 binding(HBC34-V13), while the mutation into I (HBC34-V11) partially reducedHBC34 binding, indicating that M115 could be substituted by L, but notby I, without compromising HBC34 binding. Consistently with the resultsobtained with the single mutation W107A, the double mutation W107A andM115A (HBC34-V10) completely abolished HBC34 binding.

The mutation of W107 of the VL CDR3 into F did not affect HBC34 binding(HBC34-V7), while the mutation into A (HBC34-V15) partially reducedHBC34 binding, indicating that W107 of the VL CDR3 could be substitutedby F, but not by A, without compromising HBC34 binding. As expected thecombination of the mutations W107F in the CDR3 of the VH and W107A inthe CDR3 of the VL completely abolished binding (HBC34-v2). Thecombination of the mutations M115L in VH CDR3 and W107F in VL CDR3s(HBC34-V6), of the mutations M115I in the VH CDR3 with W107F in the VLCDR3 (HBC34-v4) and of the mutations W107F in the VH CDR3 and W107F inthe VL CDR3, partially affected HBC34 binding to HBsAg, indicating thatthe combination of these two mutations, but not the individualmutations, is not compatible to retain the binding affinity of theparental HBC34 antibody.

In a next step, six of the 18 HBC34 variants described above wereselected for further characterization (HBC34-V1, V3, V4, V6, V7, V11 andV13) in order (i) to confirm the initial results; (ii) to measure thebinding affinity by ELISA (i.e. to determine the EC50 of binding); and(iii) to assess the productivity of these HBC34 variants fromtransiently transfected 293-Expi cells (FIG. 22 and FIG. 23).

As observed in the first experiment, HBC34-V3 variant (carrying thedouble mutation W107F of the VH CDR3 and W107F of the VL CDR3) bound toHBsAg (adw serotype) with an EC50 9 fold higher as compared to theparental HBC34 antibody. In addition, HBC34-V3 is produced at aconcentration almost 5 times lower as compared to HBC34. HBC34-V11 andHBC34-V13 variants carrying the single mutation M115I and M115L,respectively, bound to HBsAg with EC50 identical or slightly superior tothat of HBC34. However, both variants were produced less efficientlythan HBC34 (0.6× and 0.3× lower productivity when compared to HBC34).These results indicate that HBC34-V11, and even more HBC34-V13 variants,bind with high affinity to HBsAg but are produced less efficiently inmammalian cells. Similarly, HBC34-V1 variant carrying the singlemutation W107F in the VH CDR3 bound to HBsAg comparably with HBC34(1.6-fold higher EC50), but was produced 4-fold less efficiently (i.e.0.25×) as compared to HBC34. The combination of W107 of the VL CDR3 witheither W107, M115I or M115L of the VH CDR3 (HBC34-V3, HBC34-V4 andHBC34-V6) reduced both binding affinity (1.6-9.0 fold higher EC50) andproductivity (0.20-0.35× lower antibody concentration in the culturesupernatants). Surprisingly, the single mutation W107F of the VL CDR3(HBC34-V7) bound to HBsAg similarly to HBC34 and was produced even moreefficiently (up to 1.7×) than HBC34, reaching the remarkably highconcentration in the culture supernatant of 533 μg/ml (FIG. 23).

Example 12: Sequence Engineering of HBC34 Antibody: Framework Regions

Twelve additional HBC34 variants were produced (HBC34-V19 to HBC34-V30;FIG. 24A) in which several mutations were introduced in the frameworkregions (FRs) of both VH and VL that corresponded to the residues foundin the HBC34 unmutated common ancestor (HBC34-UCA) (FIG. 20) andcombined with the VH CDR3 mutation M115L and with the VL CDR3 mutationW107F.

Results are shown in FIG. 24. The introduction of 9 mutations in the FRsof VL (HBC34-27, HBC34-V28, HBC34-V29 and HBC34-V30) in the presence ofthe W107 mutation in the VL CDR3, combined with the WT, M115L reducedsignificantly HBC34 binding to HBsAg, thus indicating an important rolefor the mutated residues in VL (FIG. 24A-B). HBC34 variants, wherein thesame VL variant described above (i.e. W107F/FR1234-GL) was combined withthe VH carrying the M115L mutation and additional 9 mutations in FRs,did not bind to HBsAg, indicating that mutations in both VH and VLcontribute essentially to HBsAg binding.

Importantly, the removal of only one of the 9 mutations introduced inthe FRs of VL (i.e. K66Y) in HBC34-V23 and HBC34-V24 increasedsignificantly the binding (100× fold lower EC50) to HBsAg as compared tothe corresponding variants carrying the Y66K mutation (HBC34-V27 andHBC34-V28). Similarly, the removal of the K66Y mutation in HBC34-V25 andHBC34-V26 restored HBsAg binding as compared to the correspondingnon-binding variants carrying the Y66K mutation (HBC34-V27 andHBC34-V28).

Of these, the HBC34-V23 variant retained high affinity binding (1.5×higher EC50 as compared to HBC34) and was produced similarly to theparental HBC34 antibody. Of note, the HBC34-V24 variant differing foronly one amino acid from HBC34-V23 variant (i.e. M115L in VH), bound toHBsAg with a EC50 similar to that of HBC34-V23 but was not producedefficiently (only 0.14× productivity as compared to HBC34). Theseresults indicate that, while not affecting significantly the binding ofHBC34 variants to HBsAg, the presence of L at position 115 has anegative impact on the productivity of HBC34 variants carrying thismutation. Indeed, on average all HBC34 variants carrying the M115Lmutation (HBC34-V6, HBC34-V13, HBC34-V19, HBC34-V20, HBC34-V21,HBC34-V22, HBC34-V24, HBC34-V25, HBC34-V26, HBC34-V28, HBC34-V29 andHBC34-V30) have a mean productivity which is 0.3× as compared to that ofthe parental HBC34 antibody.

Remarkably, the introduction of 5 or 9 mutations in the FRs of VH in thepresence of the M115L mutation in VH CDR3 (HBC34-V19 and HBC34-V20variants, respectively) did not decrease appreciably the binding toHBsAg, suggesting that the mutated residues do not have an importantrole in the high affinity antigen recognition by HBC34 antibody. Theintroduction of the W107F mutation on the backbone of HBC34V19 andHBC34-V20 variants in HBC34-V21 and HBC34-V22 reduced the binding toHBsAg of 20-30×. Interestingly, the same mutation (i.e. W107 in the VLCDR3) did not affect the binding of other variants not carrying the same5 or 9 mutations in the FRs of the VH, a result which might indicatethat residues in the VH FRs have a cooperative role (e.g. by stabilizinga certain conformation of the variable region scaffold) in binding toHBsAg with residue 107 of the VH.

Finally and consistently with the results of Example 11 shown in FIG.23, the HMB34-V7 antibody carrying the single mutation W107 in the VLCDR3 showed a comparable binding to HBsAg (i.e. 1.4×) as compared toHBC34 and was produced more efficiently (1.2×) than HBC34 (on average inthe two experiments performed HBC34-V7 was produced 1.5×, i.e. 50%, moreefficiently than HBC34 antibody. This result suggests that the W107Fmutation in VL CDR3, while not affecting appreciably the bindingaffinity to HBsAg, has a positive impact on HBC34 antibody productivity.

Finally, HBC34 and the variants HBC34-V6, HBC34-V7, HBC34-V19, HBC34-V23and HBC34-V24 were tested for their ability to recognize the 10 HBVgenotypes A, B, C, D, E, F, G, H, I, and J (as shown in FIG. 25) by flowcytometry analysis. In particular, human epithelial cells (Hep2 cells)were transfected with plasmids expressing each of the HBsAg of the 10HBV genotypes A, B, C, D, E, F, G, H, I, and J. All antibodies weretested at multiple concentrations (8 serial dilutions from 5000 ng/ml to7 ng/ml) for staining of transiently transfected permeabilized cells.Two days after transfection, Hep2 cells were collected, fixed andpermeabilized with saponin for immunostaining with HBC34 and the fiveselected variants. Binding of antibodies to transfected cells wasanalysed using a Becton Dickinson FACSCanto2 (BD Biosciences) withFlowJo software (TreeStar). As shown in FIG. 25, HBC34 and all of thefive variants tested recognized all 10 HBV HBsAg genotypes at a similarlevel.

TABLE OF SEQUENCES AND SEQ ID NUMBERS (SEQUENCE LISTING): SEQ ID NOSequence Remarks 1 X₁X₂X₃TCX₄X₅X₆AX₇G epitopewherein X₁, X₂, X₃, X₄, X₅, X₆ and X₇ may be any amino acid 2X₁X₂X₃TCX₄X₅X₆AX₇G wherein X₁, is P, T or S, X₂ is C or S,X₃ is R, K, D or I, X₄ is M or T, X₅ is T, A or I, X₆ is T, P or L, andX₇ is Q, H or L. 3 MENITSGFLGPLLVLQAGFFLLTRILTIPQSLDSWWTSLS domain of HBsAg NFLGGTTVCLGQNSQSPTSNHSPTSCPPTCPGYRWMCL(GenBank acc. no. RRFIIFLFILLLCLIFLLVLLDYQGMLPVCPLIPGSSTTST J02203)GPCRTCMTTAQGTSMYPSCCCTKPSDGNCTCIPIPSSWAFGKFLWEWASARFSWLSLLVPFVQWFVGLSPTVWLS VIWMMWYWGPSLYSILSPFLPLLPIFFCLWVYI4 MENVTSGFLGPLLVLQAGFFLLTRILTIPQSLDSWWTS S domain of HBsAgLNFLGGTTVCLGQNSQSPTSNHSPTSCPPTCPGYRWMC (GenBank acc. no.LRRFIIFLFILLLCLIFLLVLLDYQGMLPVCPLIPGSSTTG FJ899792)TGPCRTCTTPAQGTSMYPSCCCTKPSDGNCTCIPIPSSWAFGKFLWEWASARFSWLSLLVPFVQWFVGLSPTVWLS VIWMMWYWGPSLYSTLSPFLPLLPIFFCLWVYI5 QGMLPVCPLIPGSSTTSTGPCRTCMTTAQGTSMYPSCC J02203 (D, ayw3)CTKPSDGNCTCIPIPSSWAFGKFLWEWASARFSW 6QGMLPVCPLIPGSSTTGTGPCRTCTTPAQGTSMYPSCC FJ899792 (D, adw2)CTKPSDGNCTCIPIPSSWAFGKFLWEWASARFSW 7QGMLPVCPLIPGTTTTSTGPCKTCTTPAQGNSMFPSCC AM282986 (A)CTKPSDGNCTCIPIPSSWAFAKYLWEWASVRFSW 8QGMLPVCPLIPGSSTTSTGPCKTCTTPAQGTSMFPSCCC D23678 (B1)TKPTDGNCTCIPIPSSWAFAKYLWEWASVRFSW 9QGMLPVCPLLPGTSTTSTGPCKTCTIPAQGTSMFPSCCC AB117758 (C1)TKPSDGNCTCIPIPSSWAFARFLWEWASVRFSW 10QGMLPVCPLIPGSSTTSTGPCRTCTTLAQGTSMFPSCCC AB205192 (E)SKPSDGNCTCIPIPSSWAFGKFLWEWASARFSW 11QGMLPVCPLLPGSTTTSTGPCKTCTTLAQGTSMFPSCC X69798 (F4)CSKPSDGNCTCIPIPSSWALGKYLWEWASARFSW 12QGMLPVCPLIPGSSTTSTGPCKTCTTPAQGNSMYPSCCC AF160501 (G)TKPSDGNCTCIPIPSSWAFAKYLWEWASVRFSW 13QGMLPVCPLLPGSTTTSTGPCKTCTTLAQGTSMFPSCC AY090454 (H)CTKPSDGNCTCIPIPSSWAFGKYLWEWASARFSW 14QGMLPVCPLIPGSSTTSTGPCKTCTTPAQGNSMYPSCCC AF241409 (I)TKPSDGNCTCIPIPSSWAFAKYLWEWASARFSW 15QGMLPVCPLLPGSTTTSTGPCRTCTITAQGTSMFPSCC AB486012 (J)CTKPSDGNCTCIPIPSSWAFAKFLWEWASVRFSW 16CQGMLPVCPLIPGSSTTGTGTCRTCTTPAQGTSMYPSC HBsAgCCTKPSDGNCTCIPIPSSWAFGKFLWEWASARFSW Y100C/P120T 17QGMLPVCPLIPGSSTTGTGTCRTCTTPAQGTSMYPSCC HBsAg P120TCTKPSDGNCTCIPIPSSWAFGKFLWEWASARFSW 18QGMLPVCPLIPGSSTTGTGTCRTCTTPAQGTSMYPSCC HBsAgCTKPLDGNCTCIPIPSSWAFGKFLWEWASARFSW P120T/S143L 19QGMLPVCPLIPGSSTTGTGPSRTCTTPAQGTSMYPSCC HBsAg C121SCTKPSDGNCTCIPIPSSWAFGKFLWEWASARFSW 20QGMLPVCPLIPGSSTTGTGPCDTCTTPAQGTSMYPSCC HBsAg R122DCTKPSDGNCTCIPIPSSWAFGKFLWEWASARFSW 21QGMLPVCPLIPGSSTTGTGPCITCTTPAQGTSMYPSCCC HBsAg R122ITKPSDGNCTCIPIPSSWAFGKFLWEWASARFSW 22QGMLPVCPLIPGSSTTGTGPCRNCTTPAQGTSMYPSCC HBsAg T123NCTKPSDGNCTCIPIPSSWAFGKFLWEWASARFSW 23QGMLPVCPLIPGSSTTGTGPCRTCTTPAHGTSMYPSCC HBsAg Q129HCTKPSDGNCTCIPIPSSWAFGKFLWEWASARFSW 24QGMLPVCPLIPGSSTTGTGPCRTCTTPALGTSMYPSCC HBsAg Q129LCTKPSDGNCTCIPIPSSWAFGKFLWEWASARFSW 25QGMLPVCPLIPGSSTTGTGPCRTCTTPAQGTSHYPSCC HBsAg M133HCTKPSDGNCTCIPIPSSWAFGKFLWEWASARFSW 26QGMLPVCPLIPGSSTTGTGPCRTCTTPAQGTSLYPSCCC HBsAg M133LTKPSDGNCTCIPIPSSWAFGKFLWEWASARFSW 27QGMLPVCPLIPGSSTTGTGPCRTCTTPAQGTSTYPSCC HBsAg M133TCTKPSDGNCTCIPIPSSWAFGKFLWEWASARFSW 28QGMLPVCPLIPGSSTTGTGPCRTCTTPAQGTSMYPSCC HBsAg K141ECTEPSDGNCTCIPIPSSWAFGKFLWEWASARFSW 29QGMLPVCPLIPGSSTTGTGPCRTCTTPAQGTSMYPSCC HBsAg P142SCTKSSDGNCTCIPIPSSWAFGKFLWEWASARFSW 30QGMLPVCPLIPGSSTTGTGPCRTCTTPAQGTSMYPSCC HBsAg S143KCTKPKDGNCTCIPIPSSWAFGKFLWEWASARFSW 31QGMLPVCPLIPGSSTTGTGPCRTCTTPAQGTSMYPSCC HBsAg D144ACTKPSAGNCTCIPIPSSWAFGKFLWEWASARFSW 32QGMLPVCPLIPGSSTTGTGPCRTCTTPAQGTSMYPSCC HBsAg G145RCTKPSDRNCTCIPIPSSWAFGKFLWEWASARFSW 33QGMLPVCPLIPGSSTTGTGPCRTCTTPAQGTSMYPSCC HBsAg N146ACTKPSDGACTCIPIPSSWAFGKFLWEWASARFSW 34 GRIFRSFY CDRH1 aa 35 NQDGSEKCDRH2 aa 36 AAWSGNSGGMDV CDRH3 aa 37 KLGNKN CDRL1 aa 38 EVK CDRL2 aa 39VIYEVKYRP CDRL2 long aa 40 QTWDSTTVV CDRL3 aa 41ELQLVESGGGWVQPGGSQRLSCAASGRIFRSFYMSWVR VH aaQAPGKGLEWVATINQDGSEKLYVDSVKGRFTISRDNAKNSLFLQMNNLRVEDTAVYYCAAWSGNSGGMDVWGQ GTTVSVSS 42SYELTQPPSVSVSPGQTVSIPCSGDKLGNKNVCWFQHKP VL aaGQSPVLVIYEVKYRPSGIPERFSGSNSGNTATLTISGTQA MDEAAYFCQTWDSTTVVFGGGTRLTVL 43GGACGCATCTTTAGAAGTTTTTAC CDRH1 nuc 44 ATAAACCAAGATGGAAGTGAGAAA CDRH2 nuc45 GCGGCTTGGAGCGGCAATAGTGGGGGTATGGACGT CDRH3 nuc C 46 AAATTGGGGAATAAAAATCDRL1 nuc 47 GAGGTTAAA CDRL2 nuc 48 gtcatctatGAGGTTAAAtaccgccccCDRL2 long nuc 49 CAGACGTGGGACAGCACCACTGTGGTG CDRL3 nuc 50GAACTGCAGCTGGTGGAGTCTGGGGGAGGCTGGGTC VH nucCAGCCGGGGGGGTCCCAGAGACTGTCCTGTGCAGCCTCTGGACGCATCTTTAGAAGTTTTTACATGAGCTGGGTCCGCCAGGCCCCAGGGAAGGGGCTGGAGTGGGTG GCCACTATAAACCAAGATGGAAGTGAGAAATTATATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCACTATTTCTGCAAATGAACAACCTGAGAGTCGAGGACACGGCCGTTTATTACTGCG CGGCTTGGAGCGGCAATAGTGGGGGTATGGACGTCTGGGGCCAGGGGACCACGGTCTCCGTCTCCTCA 51TCCTATGAGCTGACTCAGCCACCCTCAGTGTCCGTGT VL nucCCCCAGGACAGACAGTCAGCATCCCCTGCTCTGGAGATAAATTGGGGAATAAAAATGTTTGCTGGTTTCAGCATAAGCCAGGCCAGTCCCCTGTGTTGGTCATCTATGAGGTTAAATACCGCCCCTCGGGGATTCCTGAGCGATTCTCTGGCTCCAACTCTGGGAACACAGCCACTCTGACCATCAGCGGGACCCAGGCTATGGATGAGGCTGCCTATTTCTGTCAGACGTGGGACAGCACCACTGTGGTGTTCGG CGGAGGGACCAGGCTGACCGTCCTA 52XGSSTTSTGPCRTCMTXPSDGNATAIPIPSSWX peptidewherein the residues coded as X were substituted with Cysteines 53TSTGPCRTCMTTAQG peptide 54 GMLPVCPLIPGSSTTSTGPCRTCMTT peptide 55XSMYPSASATKPSDGNXTGPCRTCMTTAQGTSX peptidewherein the residues coded as X were substituted with Cysteines 56PCRTCMTTAQG amino acids 120-130 of the S domain of HBsAg (HBV-D J0220357 PCX₁TCX₂X₃X₄AQG, epitope wherein X₁ is preferably R or K,X₂ is preferably M or T, X₃ is preferably T or I, andX₄ is preferably T, P or L 58 QTFDSTTVV CDRL3 v7 and CDRL3 v23 (aa) 59SYELTQPPSVSVSPGQTVSIPCSGDKLGNKNVCWFQHKP VL v7GQSPVLVIYEVKYRPSGIPERFSGSNSGNTATLTISGTQA MDEAAYFCQTFDSTTVVFGGGTRLTVL 60AAGCTGGGGAACAAAAAT CDRL1 v7 and CDRL1 v23 (nuc) 61 GAGGTGAAACDRL2 v7 and CDRL2 v23 nuc 62 GTCATCTACGAGGTGAAATATCGGCCTCDRL2 long v7 and CDRL2 long v23 nuc 63 CAGACATTCGATTCCACCACAGTGGTCCDRL3 v7 and CDRL3 v23 nuc 64 TCTTACGAGCTGACACAGCCACCTAGCGTGTCCGTCTVL v7 nuc CTCCAGGACAGACCGTGTCCATCCCTTGCTCTGGCGACAAGCTGGGGAACAAAAATGTCTGTTGGTTCCAGCACAAGCCAGGGCAGAGTCCCGTGCTGGTCATCTACGAGGTGAAATATCGGCCTTCAGGAATTCCAGAACGGTTCAGCGGATCAAACAGCGGCAATACTGCAACCCTGACAATTAGCGGGACCCAGGCCATGGACGAAGCCGCTTATTTCTGCCAGACATTCGATTCCACCACAGTGGTCTTTGGC GGGGGAACTAGGCTGACCGTGCTG 65SYELTQPPSVSVSPGQTASITCSGDKLGNKNACWYQQKP VL v23 aaGQSPVLVIYEVKYRPSGIPERFSGSNSGNTATLTISGTQA MDEADYYCQTFDSTTVVFGGGTKLTVL 66INQDGSEK HBC34wt CDRH2 aa 67 EVQLVESGGGLVQPGGSLRLSCAASGRIFRSFYMSWVRQHBC34 v31, HBC34 APGKGLEWVANINQDGSEKLYVDSVKGRFTISRDNAKNSv32 and HBC34 v33 LFLQMNNLRVEDTAVYYCAAWSGNSGGMDVWGQGTT VH VTVSS 68GAGGTGCAGCTGGTGGAATCCGGCGGGGGACTGGTG HBC34 v31, HBC34CAGCCTGGCGGCTCACTGAGACTGAGCTGTGCAGCTT v32 and HBC34 v33CTGGAAGAATCTTCAGATCTTTTTACATGAGTTGGG VH (nuc)TGAGACAGGCTCCTGGGAAGGGACTGGAGTGGGTCGCAAACATCAATCAGGACGGATCAGAAAAGCTGTATGTGGATAGCGTCAAAGGCAGGTTCACTATTTCCCGCGACAACGCCAAAAATTCTCTGTTTCTGCAGATGAACAATCTGCGGGTGGAGGATACCGCTGTCTACTATTGTGCAGCCTGGTCTGGCAACAGTGGAGGCATGGACGTGTGGG GACAGGGAACCACAGTGACAGTCAGCTCC 69TCTTACGAGCTGACACAGCCCCCTAGCGTGTCCGTCT VL v23 nucCTCCAGGCCAGACAGCATCCATCACTTGCTCTGGCGACAAGCTGGGGAACAAAAATGCCTGTTGGTATCAGCAGAAGCCAGGGCAGAGTCCCGTGCTGGTCATCTACGAGGTGAAATATCGGCCTTCAGGAATTCCAGAAAGATTCAGTGGATCAAACAGCGGCAATACTGCTACCCTGACAATTAGCGGGACCCAGGCCATGGACGAAGCTGATTACTATTGCCAGACATTCGATTCCACCACAGTGGTCTTTG GCGGGGGAACTAAGCTGACCGTGCTG 70GAACTGCAGCTGGTCGAATCAGGAGGAGGGTGGGTC HBC34 wt VH codonCAGCCCGGAGGGAGCCAGAGACTGTCTTGTGCCGCA optimizedTCAGGGAGGATCTTCAGGAGCTTCTACATGTCCTGGGTGCGCCAGGCACCAGGCAAGGGACTGGAGTGGGTCGCCACCATCAACCAGGACGGATCTGAAAAGCTGTATGTGGATAGTGTCAAAGGCCGGTTCACAATTAGCAGAGACAACGCTAAAAATTCTCTGTTTCTGCAGATGAACAATCTGCGAGTGGAGGATACCGCCGTCTACTATTGCGCCGCTTGGTCTGGCAACAGCGGCGGGATGGATGTCTGGGGG CAGGGCACAACAGTGAGCGTCTCTTCC 71TCATACGAACTGACTCAGCCTCCCTCCGTCTCCGTCTC HBC34 wt VL codonACCTGGACAGACCGTCTCAATCCCCTGCTCCGGCGAT optimizedAAACTGGGCAACAAGAACGTGTGCTGGTTCCAGCACAAACCCGGACAGAGTCCTGTGCTGGTCATCTACGAGGTCAAGTATCGGCCAAGCGGCATTCCCGAAAGATTCAGCGGCTCCAACTCTGGGAATACCGCAACACTGACTATCTCTGGAACCCAGGCAATGGACGAGGCAGCTTACTTTTGCCAGACTTGGGATTCAACTACTGTCGTGTTCGGCGG CGGAACTAGACTGACTGTCCTG 72GGGAGGATCTTCAGGAGCTTCTAC HBC34 wt CDRH1 codon optimized 73ATCAACCAGGACGGATCTGAAAAG HBC34 wt CDRH2 codon optimized 74GCCGCTTGGTCTGGCAACAGCGGCGGGATGGATGTC HBC34 wt CDRH3 codon optimized 75AAACTGGGCAACAAGAAC HBC34 wt CDRL1 codon optimized 76 GAGGTCAAGHBC34 wt CDRL2 codon optimized 77 GTCATCTACGAGGTCAAGTATCGGCCAHBC34 wt CDRL2 long codon optimized 78 CAGACTTGGGATTCAACTACTGTCGTGHBC34 wt CDRL3 codon optimized 79 GGSGG linker 80 TGPCRTC epitope 81GNCTCIP epitope 82 CCIPIPSSWAFGCSTTSTGPCRTCC discontinuouswherein in particular thy cysteines at positions 2, 21, epitope mimicand 24 are coupled to acetamidomethyl. 83 CGNCTCIPIPSSWAFCSTTSTGPCRTCCdiscontinuous epitopewherein in particular thy cysteines at positions 4, 6, mimic24, and 27 are coupled to acetamidomethyl. 84 CGGGCSTTSTGPCRTCClooped epitope mimicwherein in particular thy cysteines at positions 13 and16 are coupled to acetamidomethyl. 85 STTSTGPCRTC epitope 86GNCTCIPIPSSWAFC epitope 87 GNCTCIPIPSSWAF epitope 88 PCRXC epitope

The invention claimed is:
 1. A method for treating a hepatitis B and/ora hepatitis D infection, the method comprising administering to asubject in need thereof an effective amount of an antibody, or anantigen-binding fragment thereof, that binds to the antigenic loopregion of HBsAg and neutralizes infection with hepatitis B virus andhepatitis delta virus, wherein the antibody or the antigen-bindingfragment comprises CDRH1, CDRH2, and CDRH3 amino acid sequences andCDRL1, CDRL2, and CDRL3 amino acid sequences of: (i) SEQ ID NOs: 34-38and 40, respectively; (ii) SEQ ID NOs: 34-37, 39, and 40, respectively;(iii) SEQ ID NOs: 34, 66, 36, 37, 38, and 40, respectively; (iv) SEQ IDNOs: 34, 66, 36, 37, 39, and 40, respectively; (v) SEQ ID NOs: 34-38 and58, respectively; (vi) SEQ ID NOs: 34-37, 39, and 58, respectively;(vii) SEQ ID NOs: 34, 35, 36, 37, 38, and 58, respectively; or (viii)SEQ ID NOs: 34, 66, 36, 37, 39, and 58, respectively.
 2. The method ofclaim 1, wherein the antibody or antigen-binding fragment comprisesCDRH1, CDRH2, and CDRH3 amino acid sequences and CDRL1, CDRL2, and CDRL3amino acid sequences of SEQ ID NOs: 34-38 and 40, respectively.
 3. Themethod of claim 1, wherein the antibody or antigen-binding fragmentcomprises CDRH1, CDRH2, and CDRH3 amino acid sequences and CDRL1, CDRL2,and CDRL3 amino acid sequences of SEQ ID NOs: 34-37, 39, and 40,respectively, respectively.
 4. The method of claim 1, wherein theantibody or antigen-binding fragment comprises CDRH1, CDRH2, and CDRH3amino acid sequences and CDRL1, CDRL2, and CDRL3 amino acid sequences ofSEQ ID NOs: 34, 66, 36, 37, 38, and 40, respectively.
 5. The method ofclaim 1, wherein the antibody or antigen-binding fragment comprisesCDRH1, CDRH2, and CDRH3 amino acid sequences and CDRL1, CDRL2, and CDRL3amino acid sequences of SEQ ID NOs: 34, 66, 36, 37, 39, and 40,respectively.
 6. The method of claim 1, wherein the antibody orantigen-binding fragment comprises CDRH1, CDRH2, and CDRH3 amino acidsequences and CDRL1, CDRL2, and CDRL3 amino acid sequences of SEQ IDNOs: 34-38 and 58, respectively.
 7. The method of claim 1, wherein theantibody or antigen-binding fragment comprises CDRH1, CDRH2, and CDRH3amino acid sequences and CDRL1, CDRL2, and CDRL3 amino acid sequences ofSEQ ID NOs: 34-37, 39, and 58, respectively.
 8. The method of claim 1,wherein the antibody or antigen-binding fragment comprises CDRH1, CDRH2,and CDRH3 amino acid sequences and CDRL1, CDRL2, and CDRL3 amino acidsequences of SEQ ID NOs: 34, 35, 36, 37, 38, and 58, respectively. 9.The method of claim 1, wherein the antibody or antigen-binding fragmentcomprises CDRH1, CDRH2, and CDRH3 amino acid sequences and CDRL1, CDRL2,and CDRL3 amino acid sequences of SEQ ID NOs: 34, 66, 36, 37, 39, and58, respectively.
 10. The method of claim 1, wherein the antibody orantigen-binding fragment thereof comprises a heavy chain variable domain(V_(H)) and a light chain variable domain (V_(L)), wherein V_(H) andV_(L) comprise amino acid sequences having at least 95% identity to: (i)SEQ ID NOs: 41 and 42, respectively; (ii) SEQ ID NOs: 41 and 59,respectively; (iii) SEQ ID NOs: 41 and 65, respectively; (iv) SEQ IDNOs: 67 and 42, respectively; (v) SEQ ID NOs: 67 and 59, respectively;or (vi) SEQ ID NOs: 67 and 65, respectively.
 11. The method of claim 1,wherein the antibody or antigen-binding fragment thereof comprises aheavy chain variable domain (V_(H)) and a light chain variable domain(V_(L)), wherein V_(H) and V_(L) comprise amino acid sequences having atleast 95% identity to SEQ ID NOs: 41 and 42, respectively.
 12. Themethod of claim 1, wherein the antibody or antigen-binding fragmentthereof comprises a heavy chain variable domain (V_(H)) and a lightchain variable domain (V_(L)), wherein V_(H) and V_(L) comprise aminoacid sequences having at least 95% identity to SEQ ID NOs: 41 and 59,respectively.
 13. The method of claim 1, wherein the antibody orantigen-binding fragment thereof comprises a heavy chain variable domain(V_(H)) and a light chain variable domain (V_(L)), wherein V_(H) andV_(L) comprise amino acid sequences having at least 95% identity to SEQID NOs: 41 and 65, respectively.
 14. The method of claim 1, wherein theantibody or antigen-binding fragment thereof comprises a heavy chainvariable domain (V_(H)) and a light chain variable domain (V_(L)),wherein V_(H) and V_(L) comprise amino acid sequences having at least95% identity to SEQ ID NOs: 67 and 42, respectively.
 15. The method ofclaim 1, wherein the antibody or antigen-binding fragment thereofcomprises a heavy chain variable domain (V_(H)) and a light chainvariable domain (V_(L)), wherein V_(H) and V_(L) comprise amino acidsequences having at least 95% identity to SEQ ID NOs: 67 and 59,respectively.
 16. The method of claim 1, wherein the antibody orantigen-binding fragment thereof comprises a heavy chain variable domain(V_(H)) and a light chain variable domain (V_(L)), wherein V_(H) andV_(L) comprise amino acid sequences having at least 95% identity to SEQID NOs: 67 and 65, respectively.
 17. The antibody, or antigen-bindingfragment thereof, of claim 1, wherein the antibody, or theantigen-binding fragment thereof, comprises a Fc moiety.
 18. Theantibody, or antigen-binding fragment thereof, of claim 1, wherein theantibody, or antigen-binding fragment thereof, is human.
 19. Theantibody, or antigen-binding fragment thereof, of claim 1, wherein theantibody or antigen binding fragment thereof comprises a purifiedantibody, a single chain antibody, a Fab, a Fab′, a F(ab′)2, a Fv, or ascFv.
 20. The method of claim 1, wherein the subject is receiving or hasreceived one or more of: a polymerase inhibitor; (ii) an interferon; and(iii) a checkpoint inhibitor.
 21. The method of claim 20, wherein thepolymerase inhibitor comprises lamivudine.
 22. The method of claim 1,wherein the hepatitis B infection is a chronic hepatitis B infection.23. The method of claim 1, wherein the subject has received a livertransplant.
 24. The method of claim 1, wherein the subject isnon-immunized against hepatitis B.
 25. The method of claim 1, whereinthe subject is a newborn.
 26. The method of claim 1, wherein the subjectis undergoing or has undergone hemodialysis.